Monthly Archives: October 2017

Department Press Briefings : Department Press Briefing – October 31, 2017

Heather Nauert

Spokesperson

Department Press Briefing

Washington, DC

October 31, 2017


Index for Today’s Briefing

  • BURMA
  • CUBA
  • LIBYA
  • CUBA
  • ISRAEL
  • IRAQ/ISRAEL
  • AFRICA REGION
  • IRAN/REGION
  • BURMA/REGION
  • NORTH KOREA/SOUTH KOREA/CHINA/INDIA/REGION
  • SYRIA/REGION
  • BAHRAIN
  • MIDDLE EAST PEACE

    TRANSCRIPT:


    2:23 p.m. EDT

    <a name="BURMANAT”>MS NAUERT: Hope you all are doing well today. I hope that all of you had a nice trip, some of you who went with the Secretary on his most recent trip overseas. And I know some of you are getting ready to go on the next trip, so hopefully you’re well rested and that none of you will be too grumpy to turn off the lights and not answer the door for trick-or-treaters tonight. (Laughter.) Because those folks are never fun, the ones who pretend they’re not home.

    In any event, I’d like to start with a couple things today. First is the issue of Burma and I want to announce some travel that is underway right now. Simon Henshaw, who’s our Acting Assistant Secretary of State for the Bureau of Population, Refugees, and Migration, is currently leading a U.S. delegation to Burma and Bangladesh to discuss the humanitarian and human rights concerns stemming from the Rakhine State crisis. The delegation is close to completing the first leg of its trip in Burma. They have met with Rohingya and Rakhine State community leaders, including a visit to a camp for those who’ve been internally displaced, as well as holding meetings with government and civil society members.

    The delegation will next travel to Bangladesh, where they will have the chance to visit affected communities in Cox’s Bazar. Cox’s Bazar, for those of you who do not know, is an enormous refugee camp that is serving as temporary home, we certainly hope – to somewhere between 800,000 and a million refugees. About 600,000 of them have come from Burma alone, so it is an enormous place.

    The Acting Assistant Secretary Henshaw is accompanied by the bureau – excuse me – by the Deputy Assistant Secretary Scott Busby of the Bureau of Democracy, Human rights, and Labor. Those of you who were here last week had the chance to hear from Mr. Busby, who spoke about the DPRK and human rights abuses there. Also joined – they are joined by Acting Deputy Assistant Secretary Tom Vajda of the Bureau of SCA and Office Director Patricia Mahoney of the Bureau of East Asian and Pacific Affairs.

    Secondly, I want to talk about Cuba for a second. As many of you are probably aware, tomorrow is the annual Cuba embargo resolution vote at the United Nations. For 26 years, Cuba has introduced a resolution for the General Assembly, calling for the end to the U.S. embargo on Cuba. We’ve historically voted against that resolution. Last year the United States abstained. And Matt, if I’m not mistaken, you and the AP broke that story last year.

    QUESTION: We – I think so, yeah.

    MS NAUERT: You think so. So many you can’t remember. (Laughter.) Okay. Well, a little bit of news for you on that front. Ambassador Haley will be reversing last year’s abstention and will vote against the resolution this year. It will be the first vote since President Trump announced our new Cuba policy. We plan to vote against the resolution to underscore this new approach to Cuba. The Trump administration policy gives greater emphasis in advancing human rights and democracy in Cuba, while maintaining engagement that serves U.S. national interests, maintains engagement on areas of U.S. national interest, ensures U.S. engagement benefits the Cuban people, and ensures compliance with the statutory ban on tourism to Cuba.

    And lastly, I’d like to mention something that was incredibly important to many people who serve here at the State Department. Yesterday, as many of you are aware, the White House announced the arrest of Mustafa al-Imam, one of the perpetrators of the September 11, 2012 attack on our consulate in Benghazi, Libya that killed four Americans, including two of our State Department colleagues. The two State Department colleagues, of course, were Ambassador to Libya Chris Stevens and also Sean Smith.

    Many of you here know, as you walk into the foyer of our building, you will see the names of those who served the State Department who died in service of the State Department and the American people. Those who’ve not been to this building in Washington before, it is an incredible sight to see the names of those who have died in service of our country.

    On behalf of the State Department, we would like to express our gratitude to the U.S. military, to law enforcement, to the Intelligence Community for their relentless efforts to bring to justice the perpetrators of those attacks. Today, I spoke with some of my colleagues who served with these men. They described the news of the arrest as, quote, “emotional,” saying that it marked an important day. They told me that the memory of Ambassador Stevens and the other men drives them and provides an ongoing tribute to the work that they are still engaged in in Libya and around the issues involving Libya today.

    I’m reminded of a conversation that I had with Admiral Mike Mullen, the former chairman of the Joint Chiefs of Staff, just before I took this position. He said to me about Ambassador Stevens, that he died doing exactly what an ambassador should be doing – serving in the field, working with locals in places that are not always safe. And I’ve never forgotten what Admiral Mullen said to me. It is a good reminder that the 75,000 people who work for the State Department around the world often serve in dangerous locations and they make tremendous sacrifices on behalf of our country. We continue to mourn the loss of Ambassador Chris Stevens, Glen Doherty, Sean Smith, and Tyrone Woods, and we will spare no effort to ensure that justice is served for these dedicated Americans and public servants. Our colleagues today continue the work that they started not only in the same spirit, but also in their honor.

    Thank you. I’d be happy to take your questions.

    QUESTION: Thanks, Heather. Let’s start with the Cuba vote. The Secretary and other senior members of this administration have repeatedly said that the Trump administration’s slogan, “American First,” does not mean America alone. I’m sure you’re aware of the previous years’ votes in the UN General Assembly on the Cuba Embargo Resolution. It’s 191 to 2, you and Israel being the only ones to vote against it, or, as in the case last year, you both abstained. You lost the Marshall Islands, Micronesia, Palau in 2012 to 2013. How is this not America alone?

    MS NAUERT: Look, I think when we talk about America First, this is a global issue where we’re putting Americans first and the concerns and interests and the safety of secure – and security of American people first. I think this administration regard – would regard that for far too long Cuba has engaged in human rights abuses, human rights abuses that perhaps past administrations have turned and looked the other way, and this administration continues to call upon Cuba to improve – to improve in terms of human rights, and also open up to where they would have better media access, better access to the things that we enjoy here.

    QUESTION: Do you – does this decision have anything to do with the attacks that have happened on – that you guys have talked about?

    MS NAUERT: No, no. Look, we undertook – this administration undertook a very broad Cuba policy review, something that the President outlined and underlined and underscored earlier this year. So I’d just refer you back to what – the President’s comments on that, but that is where things stand.

    QUESTION: No, but I mean, the decision on this – the decision to vote against this resolution doesn’t have anything to do with those —

    MS NAUERT: No. That would be a separate matter altogether.

    QUESTION: All right. And then just lastly, have you talked to the Israelis? Are they going to vote with you again?

    MS NAUERT: I have not spoken to the Israeli Government about this.

    QUESTION: Well, do you know if any – anyone has?

    MS NAUERT: I don’t know that answer offhand.

    QUESTION: Okay. And then just the last thing on this. Does it – the United States’ position as well as Israel’s position on boycotts of countries is that they are – particularly when it comes to Israel, is that they are opposed to them. Is it still the administration’s policy that boycotting countries such as the BDS movement is something that you oppose?

    MS NAUERT: Do we oppose —

    QUESTION: Boycotts of Israel?

    MS NAUERT: Boycotts of Israel. Yeah. I mean —

    QUESTION: I’m just trying to figure out if you see —

    MS NAUERT: Look, we’ve —

    QUESTION: If you see that there’s a —

    MS NAUERT: We’ve had an example where there have been blacklists that have put – been put together of companies —

    QUESTION: Right. I just want to —

    MS NAUERT: — that have been working in what some would consider to be disputed areas. We tend to think that that is not a good idea.

    QUESTION: Right. I just want to make sure that I understand correctly. The administration does not see it as inconsistent to support its embargo on Cuba, which is opposed by every other country in the world with the exception of one, and its opposition to embargoes or boycotts of Israel? That’s not inconsistent, right?

    MS NAUERT: We look at the Cuban Government and some of the activities that it’s done, some of the things that it’s done to its very own people, and see that as problematic. The President believes very firmly in focusing on human rights issues in Cuba among any other things, and I think that that is a testament to the tremendous value that we place on human rights and dignity. And other countries don’t want to call out nations like Cuba for that kind of activity, but we do.

    QUESTION: I get it. But you don’t think that – you don’t see an inconsistency in the position?

    MS NAUERT: I don’t. No.

    QUESTION: Okay. All right.

    MS NAUERT: Okay.

    QUESTION: Thank you.

    MS NAUERT: All right. Where would we like to go? Good afternoon. Laurie, do you want to start with Iraq today?

    QUESTION: Iraq. Iraq. Okay.

    MS NAUERT: Okay.

    QUESTION: And I have several questions. The Iraqi Government seems to have a campaign against journalists. A Kurdistan TV reporter, Arkan Sharif, was killed yesterday in Kirkuk apparently by hostile Shaabi. Today, the prime minister said Kurdish channels were guilty of war crimes just by reporting on the fighting, and Kurdistan 24 and other Kurdish channels have been banned in areas of Iraq that are controlled by Baghdad. And an Arab journalist – so it’s not just Kurds – Samir Obeid, who was critical of Abadi, was arrested. What’s your comment on all that?

    MS NAUERT: Yeah. I have not heard of all of that, Laurie. I’m aware of the murder that took place of the journalist in Kirkuk. We’ve seen that story. Some of this information is just coming in to us, so I can’t confirm all of these stories that you’re mentioning. We are certainly aware of media reports based in the – based on the government having issues with the central government of Iraq. And media issues overall – I mean, our position has not changed. We support freedom of the press. We believe that more voices, not fewer voices, is good for democracy, is good for people of various countries. We would mourn the arrest – I mean, excuse me. We would mourn the death of any journalist covering this, trying to bring additional information to the people of Iraq.

    QUESTION: The Committee to Protect Journalists has condemned this. Do you expect if these things continue you will also be condemning this?

    MS NAUERT: Yeah, I think that’s just – that’s a hypothetical, so I don’t want to get ahead of anything. But certainly, that would be a concern of ours if there are attempts to squash the voices of those who are trying to just bring more information to the people.

    QUESTION: My next question has to – is a continuation of our discussion last time, when you Abu Mahdi al-Muhandis was a terrorist. There’s another Hashd al-Shaabi commander, Qais al-Khazali, a prominent figure who was detained at Camp Cropper for killing U.S. troops during Operation Iraqi Freedom. And the Iraqi Interior Minister Qasim al-Araji, has longstanding ties to the IRGC, whom you just named as a terrorist organization, and he was also detained by the U.S. during OIF. What is your comment on these people? Are they terrorists, in your view?

    MS NAUERT: Yeah, Laurie, I don’t want to get too much into that because our focus is on trying to get Iraqis, the Kurds to come together and have some sort of dialogue. I fully understand and appreciate your question. I know well who those men are that you speak of. We are very familiar, as the United States Government is, in the acts that they are believed to be responsible for. And that, of course, is a tremendous concern to us.

    You mention Mr. Muhandis. He was designated by the Department of Treasury back in 2009 for threatening the peace and stability of Iraq and also the Government of Iraq. We are well aware of that, but I just don’t want to get too into the details of what you’re asking.

    QUESTION: Let me give you a softball question then. The Iraqi parliament —

    QUESTION: You can’t get better than that.

    MS NAUERT: Yeah, right. Thanks.

    QUESTION: Okay.

    QUESTION: (Off-mike.)

    QUESTION: The Iraqi parliament voted today to criminalize the display of the Israeli flag and what it called other Zionist symbols inside Iraq. What is your comment on that one?

    MS NAUERT: Yeah. I mean, look, you know very well that we have a close relationship with the Government of Israel. That, however, would be an internal Iraqi matter.

    QUESTION: You’re not going to condemn this sort of thing?

    MS NAUERT: Look, if they choose to do that, that is certainly their place to do that. That would be an internal Iraqi matter. But I think the Israelis very well know how much we support them and what a strong relationship we have with the Government of Israel. Okay?

    QUESTION: New topic?

    QUESTION: Heather?

    MS NAUERT: Okay.

    QUESTION: New topic. On the KRG, Heather.

    MS NAUERT: Okay.

    QUESTION: Right. Just a follow-up on some of the Kurdish question. We saw the statement yesterday regarding Barzani’s announcement to step down. Now, as Washington is encouraging the reconciliation process between the Iraqis and Kurds, what role does the United States play in the negotiation process? As a facilitator, or does the U.S. play any role at all? Thank you.

    MS NAUERT: Well, we’ve had a series of conversations previously with Mr. Barzani, as well with Prime Minister Abadi. The Secretary has had conversations with both men. Our ambassador has been extremely engaged not only with the Kurds, but with the Iraqi regional government. Our ambassador has had lots of conversations with people on the ground. As you know, we have a lot of people who are serving there as well.

    So those conversations continue. We would like both parties to sit down and have a dialogue together, and we’re hopeful that they’ll eventually be able to work it out.

    QUESTION: Now, does the United States still consider the Kurds as a good ally in the fight against ISIS in Iraq?

    MS NAUERT: Absolutely, without a doubt. I mean, we are grateful for the hard work and the bravery of the Kurdish Peshmerga. That has not changed. We’ve always believed that, and we will still consider them to be a strong fighting force. Okay?

    QUESTION: And finally, could you please address some of the questions from the Kurdish people that some heavy weapons provided by the United States are used by the Iraqi armies to use against the Kurds?

    MS NAUERT: So as you all know, we have provided weapons to the Iraqi Government – let me see where – pardon me one second. Yeah, I mean, we’ve certainly trained, as you well know, alongside and with the Kurds and the Iraqi regional government. We don’t provide – perhaps there’s been some questions about this. We don’t provide support to groups or forces that are designated terror organizations – I think we’ve been very clear about that – some believed to be responsible for gross violations of human rights that do not fall under the government – under the control of the Government Iraq. Okay?

    QUESTION: Is it a violation of U.S. regulations if a – an entity that to whom you have provided weapons transfers those weapons to a terrorist organization?

    MS NAUERT: I know there’s something called the end-use law, I believe it is, which regulates what different parties are allowed to or not allowed to do with weapons that the U.S. provides that. Some of that is a DOD – in DOD’s lane, so I don’t want to get too much into that, because I’m not an expert on the matter, but I know that there are those guidelines that are put into effect.

    QUESTION: Maybe that’s something we could pursue either here or at the State Department or with the Department of Defense?

    MS NAUERT: You’re certainly welcome to ask the – I’ve seen you over at the Department of Defense in the Pentagon briefings before.

    QUESTION: Yes (inaudible).

    MS NAUERT: You’re certainly welcome to ask them that question. I can see if we can get anything more for you on that.

    QUESTION: Okay. Thank you very much.

    QUESTION: Question on the Sahel?

    MS NAUERT: Okay. Would you like to move on now?

    QUESTION: The Sahel?

    MS NAUERT: Okay. Sorry?

    QUESTION: The Sahel?

    MS NAUERT: Yes.

    QUESTION: The Secretary announced yesterday $60 million for this new G5 force.

    MS NAUERT: Mm-hmm.

    QUESTION: Can you explain a little bit about what that money is going to go to? And is that – is the U.S. cutting back on kind of aid to that area, with the big cutbacks of this administration?

    MS NAUERT: The dollar amount that was announced yesterday, a total of $60 million for the G5 countries – those G5 countries include Burkina Faso, Chad, Mali, Mauritania, and Niger – the point for that money is to try to create a joint force and also recognize that they are making progress toward operating better military and counterterrorism operating capability. Of the $60 million, not all of it comes from the State Department; $45 million comes from the State Department, 15 million comes from DOD. That, added up, is $60 million. That is being given through peacekeeping operations, so it’s not going through the United Nations; it’s going from the State Department directly to some of them. So I know the Secretary was pleased to be able to announce that kind of level of cooperation.

    Okay. Anything else on that?

    QUESTION: There were existing DOD programs in that area. Is this entire 60 million new funding? Obviously, there’s been train-and-assist missions in there for several years. Do you know whether this —

    MS NAUERT: Well, I can’t —

    QUESTION: — replaces anything, or whether it’s 60 million on top of existing bilateral figures?

    MS NAUERT: I can’t speak to the DOD’s $15 million contribution to this. I believe that this is a new pocket, or a new pot of money that is going to them, but I can double check on that. Okay?

    Anything else on this issue? Okay.

    QUESTION: (Off-mike.)

    QUESTION: Iran.

    MS NAUERT: What do you got? Iran, okay.

    QUESTION: Hi, Heather. Thanks. Iran’s supreme leader says that he is going to put a limit on the ballistic missile program in his country to 2,000 kilometers. Does the United States view that as a concession? Does that change U.S. policy or posture at all towards —

    MS NAUERT: Well, 2,000 kilometers is actually pretty far. That would certainly put other countries that are allies of ours in the range. I’m not going to comment on every statement that comes out from an Iranian official, nor any other government official from around the world, for that matter. We all know, as do our partners in the region – they know, certainly, that Iran’s ballistic missiles threaten not only the United States but our partners in the region, including many Arab countries. They recognize that. We have plenty of conversations with those governments and those countries about their concerns for Iran’s destabilizing activity. So it’s not just the United States that considers Iran to be a threat in that regard; it’s our partner nations as well.

    Those Iranian-made rockets have too long been used to exacerbate some of the – and inflame conflicts in the region. And as you well know, it’s in violation, ballistic missiles are, of the UN Security Council resolutions.

    QUESTION: Despite the range, the U.S. wants to see a range of zero, I assume, then, still?

    MS NAUERT: We would like to see them come into compliance with the UN Security Council resolutions, and would see these in violation of the UN Security Council resolutions.

    QUESTION: (Sneeze.)

    QUESTION: Bless you.

    MS NAUERT: Bless you. Okay. Where would we like to go now?

    QUESTION: Iran?

    MS NAUERT: Okay.

    QUESTION: So administration officials have confirmed that Tillerson floated the idea of top U.S. Government officials meeting with Iranian Government officials during the UN General Assembly meetings. Can you give us some more details on that?

    MS NAUERT: Yeah. So I know that that was something that the Secretary had floated. The country – the Iranian officials said no, and that was the end of it.

    QUESTION: Well, wait a second. The Secretary is the most senior-ranking cabinet agency chief in this government. He met with the Iranian foreign minister. You’re talking about something other than that?

    MS NAUERT: Well, they had the meeting in New York, which was a part of —

    QUESTION: I know, and they sat in a room alone with their delegations for several minutes.

    MS NAUERT: Which was a part of – the countries that are part of the signatory to the JCPOA. And they had a lengthy conversation, and the Secretary certainly called out, as many of you have probably read, the Iranian Government for its destabilizing activities that date back 40-plus years.

    QUESTION: Yeah, I’m not – my question is – her question was: Did he raise the idea of senior officials meeting with Iranian officials? And you’re saying —

    MS NAUERT: I think I just answered that.

    QUESTION: Yeah, but he himself met with a senior Iranian official.

    MS NAUERT: Yes.

    QUESTION: Are you talking about he floated the idea of other administration officials meeting with the same Iranian official he met or even more senior Iranian officials?

    MS NAUERT: I’m not aware of exactly who it was, who in the Iranian delegation would have been included in that, but I know that that idea was floated.

    QUESTION: And floated with whom? With Zarif?

    MS NAUERT: I’m not sure exactly.

    QUESTION: And so was Tehran correct in saying that Iran had turned down the offer for a meeting with the President Trump?

    MS NAUERT: They did.

    QUESTION: Okay.

    QUESTION: They did turn down a meeting with the President?

    MS NAUERT: They did turn down a meeting, yes, with U.S. officials.

    QUESTION: With —

    MS NAUERT: I believe —

    QUESTION: With President Trump.

    MS NAUERT: I believe the United – I believe that the White House commented on this yesterday —

    QUESTION: Did they? Okay.

    MS NAUERT: — so I’d refer you back to the White House for some of that, okay?

    QUESTION: They did?

    MS NAUERT: I believe they did.

    QUESTION: I wasn’t aware there were any questions —

    QUESTION: I don’t think that they did.

    QUESTION: — at the briefing other than certain legal activity that’s going on.

    MS NAUERT: Okay.

    QUESTION: Yeah.

    MS NAUERT: Then you should’ve been over there talking about other things.

    Okay, shall we move on?

    QUESTION: (Off-mike.)

    MS NAUERT: Okay. Okay, yeah.

    QUESTION: North Korea?

    QUESTION: Just a quick follow-up on that.

    MS NAUERT: Okay.

    QUESTION: When you said that the delegation has met with government officials, does that include military officials?

    MS NAUERT: Not to my awareness. I can only speak on behalf of the State Department, the State Department with our various bureaus – democracy, labor, human rights; EAP, East —

    QUESTION: Burmese military officials.

    MS NAUERT: Will we be meeting with Burmese military officials? I don’t believe so. Let me just check my notes. I know that the Secretary has had conversations with the–General Min from Burma. I don’t have a conversation to read out to you, but we have certainly had conversations with them, as we have Aung San Suu Kyi and many others in the government as well, so —

    QUESTION: Can you take the question, I guess, whether —

    MS NAUERT: Yeah. Let me just try to find – see what information I have for you that I can provide.

    QUESTION: Just while we’re on Burma, Myanmar, you said he’d been able to meet with – the envoy has managed to meet with the Rohingya. Has he been able to go everywhere in the country he’d like – would have liked to have done? I know before there was —

    MS NAUERT: I’m sorry, what did you say?

    QUESTION: Has he been able to travel wherever within Burma he wanted to go? I know there’s been restrictions in the past on access.

    MS NAUERT: Yeah. So let me give you a little bit of information on where he’s at – and Conor, I’m just finding it right now. So our delegation traveled to Sittwe, to Rangoon, and also to Naypyidaw.

    QUESTION: Naypyidaw.

    MS NAUERT: Naypyidaw. Thank you, Matt. They – the diplomatic mission’s primary purpose was to work with the government and other actors – I know we’ve been speaking with various aid groups in the area to learn about their experiences in trying to get equipment, material, and other humanitarian-type programs to the people of Burma who are suffering. There have been, as many of you know, more than 600,000 Rohingya who have been forced to leave their areas. Many of them have wound up in the neighboring Bangladesh, and I believe it’s tomorrow or the following day where Acting Assistant Secretary Henshaw will be in Bangladesh to take a look at – meet with some people in conjunction with their time in Bangladesh. Okay?

    QUESTION: Can I just ask one more question?

    MS NAUERT: Yeah.

    QUESTION: Is it the administration’s view that it’s better to work with the Burmese Government to help solve the crisis as opposed to beginning to put some pressure on the government?

    MS NAUERT: Well, we’ve certainly put pressure on the government, without a doubt. I mean, many of you saw the sanctions that were released late last week. It was pretty lengthy. So I think we view this as a multi-pronged approach. Part of that is talking with the government, having conversations, but also recognizing that the government is a fledgling democracy, that they need help. Certainly, we have been very clear about expressing our severe concerns. You are all aware of the conversations that we’re having internally about the situation that has befallen the Rohingya. You also know about many travels that our team has made to the region to meet with people and get more information on the ground there. Okay?

    QUESTION: So additional sanctions would be —

    MS NAUERT: You know I’m not going to forecast any potential additional sanctions.

    QUESTION: Sure.

    MS NAUERT: But that – those things are always an option. Okay?

    Hey, Laura.

    QUESTION: Hey. Could we go to North Korea?

    MS NAUERT: Sure.

    QUESTION: Is the State Department going to relist North Korea as a state sponsor of terrorism?

    MS NAUERT: So our Bureau of Legislative Affairs talked with us a little bit earlier today about the deadline that we have for that. There’s a congressionally mandated deadline. We’ve been working under the deadline of November the 2nd. That is something that we are certainly looking at. We’ve been looking at this for quite some time, but I just don’t have anything to announce for you today.

    QUESTION: Why November 2nd instead of today?

    MS NAUERT: I’m getting that from our leg. affairs people. Okay? Okay.

    QUESTION: Heather, real quickly —

    MS NAUERT: Yeah.

    QUESTION: — has anybody at State spoken to the family of Otto Warmbier about this?

    MS NAUERT: I know we’ve spoken with their family in the past. I’m not sure if we’ve spoken with them in recent days.

    QUESTION: About this specifically?

    MS NAUERT: I’m not aware that we have. Okay?

    QUESTION: Heather.

    MS NAUERT: Yeah, hi.

    QUESTION: You want to go —

    QUESTION: The same issue, South – North Korea —

    MS NAUERT: Yeah, DPRK?

    QUESTION: Yeah.

    MS NAUERT: Okay.

    QUESTION: South Korea abstained from a UN resolution condemning North Korea’s nuclear test. In this regard, South Korea rejected these issues. It seems to be not helpful for the UN sanctions against North Korea.

    MS NAUERT: I’m sorry, Janne. Could you start that question over again, please?

    QUESTION: South Korea abstained from —

    MS NAUERT: South Korea abstained?

    QUESTION: Yes – the UN resolution condemning North Korea’s nuclear test recently – last week, I think, that happened – United Nations.

    MS NAUERT: I’m sorry. I’m not aware of that UN resolution vote, so I would have to refer you, then, to our —

    QUESTION: Condemning North Korea’s nuclear test (inaudible).

    MS NAUERT: I’m not aware of that vote having taken place. I’d just have to refer you to our USUN staff on that.

    QUESTION: All right. Thank you.

    MS NAUERT: Okay. Hi.

    QUESTION: Can I ask a related – somewhat related question? Yes? Or —

    MS NAUERT: Sure, yeah, yeah.

    QUESTION: Okay. Thank you. What’s your understanding of China’s position on the deployment of THAAD in South Korea right now?

    MS NAUERT: Yeah. I think that China recognizes, they certainly do – well, first of all, let me start by saying we certainly welcome that China and the Republic of Korea would have a closer relationship. We tend to think that that is a good thing for the region and especially the regional instability and the worldwide instability that the DPRK poses, the threat that they pose.

    In terms of THAAD, nothing has changed from our position on that. It is a – was an alliance decision on the part of the U.S. and the Republic of Korea, something that we came to together. As you well know, it’s a defensive mechanism; it’s not something that’s offensive. One of our priorities is not only keeping our own people safe, but keeping our allies safe.

    QUESTION: Yeah, but that’s – I know why —

    MS NAUERT: Yeah.

    QUESTION: — what your position on THAAD is.

    MS NAUERT: Yeah, okay.

    QUESTION: I’m wondering what your position is on China’s position on it.

    MS NAUERT: Our position —

    QUESTION: Are you pleased – are you pleased that the Chinese seem to have kind of overcome their just absolute complete hostility toward – to it to the point where they’re ready to start high-level contacts again with the South Koreans and ease the tensions between the two?

    MS NAUERT: And Matt, that’s just what I said. We would certainly welcome and we’re pleased to hear that the Republic of Korea, that our Korean friends and also the Chinese are forging a closer relationship. We see that as providing better stability, greater stability for a region that desperately needs it because of North Korea.

    QUESTION: Is it your understanding that some of that – the rapprochement between the two is somewhat related to the Chinese dropping their extreme objection to THAAD?

    MS NAUERT: I don’t know.

    QUESTION: All right.

    MS NAUERT: I don’t know. I can’t answer that.

    QUESTION: Heather —

    MS NAUERT: Okay.

    QUESTION: — just to follow up, do you see this as an indication that China’s recognizing that North Korea is a strategic liability and not an asset?

    MS NAUERT: I think that China is coming even closer to recognizing that North Korea is a thorn in its side, and a thorn in the side of many nations. Obviously, they have a lot of trade that goes through, flows through North Korea. But it is not without significance that the Chinese backed two UN Security Council resolutions calling out North Korea for its destabilizing activities. So China, I think, is certainly coming around and recognizing the threat that the DPRK poses. Okay? Okay.

    QUESTION: Can I follow up? So while the United States is asking for cooperation on North Korea issue – but Secretary Tillerson last week gives a speech which he envisioned the U.S.-India relation for the next century, and in that speech he criticized China on many fronts like South China Sea and economic expansion. So many experts see this as a China containment strategy. Could you please clarify on it? Is it a China containment strategy?

    MS NAUERT: I think the Secretary and what he said in his speech about China, in his speech about India, was something that the Secretary has said with China privately before. So some of those in the past have been private conversations, and now they’re just becoming more public conversations. But let me just say I know that the President is very much looking forward to his trip to China. It is going to be a lengthy trip, a robust trip, and one of the top issues that will be discussed with China is certainly the DPRK.

    QUESTION: But if the United States —

    QUESTION: (Off-mike.)

    QUESTION: — values a strong India-United States relation, why India is not included in the first – President Trump’s first Asia trip?

    MS NAUERT: I think that that would be a different kind of trip for the President, tagging India on – along to that trip. He’s got a pretty hefty schedule, but I don’t want to speak on behalf of the White House.

    QUESTION: Can I have a follow-up (inaudible)?

    QUESTION: Well, does that mean Secretary Tillerson and Secretary Trump – Secretary Tillerson and President Trump think differently on India strategy?

    MS NAUERT: No. I mean, they have the same position on that, the same – the same concerns, the same foreign policy goals and all of that, but I’m not going to get ahead of the President’s trip and why certain countries are on his schedule and why others are not. That would be under the White House’s purview. Okay.

    QUESTION: Can I have a follow-up?

    MS NAUERT: Okay. Hi.

    QUESTION: Yes. And just in Tillerson’s speech, he also talked about more involvement of the United States for the infrastructure investment in the Asia Pacific and more connectivities for that region. So can we say that this is kind of a U.S. substitution for China’s Belt and Road Initiative? Or – he also talked about an agreement between the U.S. and Nepal. Is there any other agreements or – or the negotiations that is going on you could just – you could just tell us?

    MS NAUERT: Yeah. I can’t forecast any agreements or negotiations, especially nothing before a trip.

    QUESTION: And about the Thousand Girls Initiative, do you think that’s a U.S. version of that, so there’s a competition?

    MS NAUERT: I can tell you – I can tell you that we have a close relationship with India, that we have a lot of areas of common interest including we’re both democracies, we’re both large countries. They are an enormous country. India can bring so much not only to the region but to the world. In addition, many American jobs through greater trade and cooperation with that country, and I’m just not going to get beyond that. The Secretary gave a very lengthy speech on India and I can just refer you back to the text of that speech. Okay? All right.

    Go right ahead.

    QUESTION: Thank you. I have a question on Syria. Yesterday Secretary Tillerson, he said, quote, that, “We are working on establishing additional de-escalation zones in Syria.” So what are the areas, and are they, like, the de-escalation zone that was established in July with Russia and Jordan?

    MS NAUERT: Yeah. So we’ve talked about this here before, that there is an area where the United States and Russia have had an area of mutual interest, and that is to try to bring some peace and some stability to parts of Syria. The goal would be to build upon that, to create some better trust between our countries, and to be able to develop other areas where we could have a ceasefire.

    The ceasefire that you’re talking about, in other words de-escalation zone, has been in effect since July the 9th. That’s held pretty well, and that has been successful. We’ve been successful in being able to help get humanitarian aid and other supplies into that area. If we can build upon that in other regions, then that would be a move that we would very much support. In terms of the Secretary’s conversations in particular about that within the past few days, I don’t have anything for you on that.

    QUESTION: Work is going on now according to Secretary Tillerson.

    MS NAUERT: I can’t confirm that that work is going on, but I know that that remains a shared goal, at least on the part of our administration, that we would like to see that, because ultimately we’d like to see peace and stability, and we’d like to see so many Syrians be able to come back home. Okay? We’re going other have to wrap it up, guys.

    QUESTION: All right, Heather?

    MS NAUERT: Yeah.

    QUESTION: I have two very brief ones, both on the Middle East. First on Bahrain.

    MS NAUERT: Okay.

    QUESTION: Yesterday, a Bahraini court sentenced three relatives of a U.K. British-based human rights activist to three years in prison for various insults. Both – this was condemned by both Amnesty International and Human Rights Watch, which is not surprising, but I’m just wondering if you guys have any comment on it as well since you’ve been quite outspoken on other Bahraini – other cases in Bahrain about this.

    MS NAUERT: Yeah. So you’re referring to Bahraini activist Mr. Sayed Nazar Alwadaei. So we understand and we’re certainly aware that he was sentenced to three years in prison along with some family members who are living in Bahrain, but he’s based in London, according to my understanding. He alleges that the convictions on the part of the Bahraini Government were reprisals for his activities in London. That I can’t – I can’t personally confirm that. But we understand that there are also allegations that he confessed under duress.

    We always will say this, that we urge the Bahraini authorities to investigate allegations thoroughly and also impartially. And I just don’t have anything more for you on that.

    QUESTION: Okay. And then lastly, and I’ll do this because Said is not here.

    MS NAUERT: Where is Said?

    QUESTION: It wouldn’t be a question – it wouldn’t be a briefing if there wasn’t an Israel-Palestinian question.

    MS NAUERT: Okay.

    QUESTION: So on Sunday, Prime Minister Netanyahu decided not to have a vote on annexing some of these settlements to the city of Jerusalem, and the reason that he gave was that the U.S. may be presenting a peace plan and he doesn’t want to do anything without consulting the – in terms of this law without consulting the U.S. What’s he talking about?

    MS NAUERT: I think we would certainly like to see a peace plan.

    QUESTION: Yes. But are you getting ready to propose one?

    MS NAUERT: Our people have been – our people have been very hard at work with a lot of trips over to the region. As we have said many times before, there will be a lot of trips. There will be a lot of trips to the region, a lot of meetings, and all of that, but ultimately, for any kind of peace agreement to work, both sides have to be willing to agree to it. It can’t be something that we impose; both sides have to be willing to – and able to live with it.

    QUESTION: Right. But he seemed to suggest that something might be imminent. Are you aware of anything that —

    MS NAUERT: I’m not. I don’t have any – I don’t have anything to announce.

    QUESTION: All right, thank you.

    MS NAUERT: All right, thanks everybody. I’ll see you later.

    (The briefing was concluded at 2:59 p.m.)



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An Investigation of the Single and Combined Phthalate Metabolite Effects on Human Chorionic Gonadotropin Expression in Placental Cells

Author Affiliations open

1Department of Epidemiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA

2Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

3Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California, USA

4Department of Clinical Chemistry, Biomedicum, Helsinki University and Helsinki University Central Hospital, Helsinki, Finland

5Institute of Reproductive and Developmental Biology, Imperial College of London, London, United Kingdom

PDF icon PDF Version (1.6 MB)

  • Background:
    Observational studies have reported associations between maternal phthalate levels and adverse outcomes at birth and in the health of the child. Effects on placental function have been suggested as a biologic basis for these findings.
    Objective:
    We evaluated the effects of phthalates on placental function in vitro by measuring relevant candidate genes and proteins.
    Materials and Methods:
    Human trophoblast progenitor cells were isolated at 7–14 wk of pregnancy (two female and three male concepti), and villous cytotrophoblast cells (vCTBs) were isolated at 15–20 wk (three female and four male concepti). Cells were cultured in vitro with four phthalate metabolites and their combination at concentrations based on levels found previously in the urine of pregnant women: mono-n-butyl (MnBP, 200 nM), monobenzyl (MBzP, 3 μM), mono-2-ethylhexyl (MEHP, 700 nM), and monoethyl (MEP, 1.5 μM) phthalates. mRNA levels of CGA, CGB, PPARG, CYP19A1, CYP11A1, PTGS2, EREG, and the intracellular β subunit of human chorionic gonadotropin (hCGβ) and peroxisome proliferator activated receptor γ (PPARγ) were measured in the cellular extracts, and protein levels for four forms of secreted hCG were measured in the conditioned media.
    Results:
    Previously reported associations between maternal phthalates and placental gene expression were reproduced experimentally: MnBP with CGA, MBzP with CYP11A1, and MEHP with PTGS2. CGB and hCGβ were up-regulated by MBzP. In some cases, there were marked, even opposite, differences in response by sex of the cells. There was evidence of agonism in female cells and antagonism in male cells of PPARγ by simultaneous exposure to multiple phthalates.
    Conclusions:
    Concentrations of MnBP, MBzP and MEHP similar to those found in the urine of pregnant women consistently altered hCG and PPARγ expression in primary placental cells. These findings provide evidence for the molecular basis by which phthalates may alter placental function, and they provide a preliminary mechanistic hypothesis for opposite responses by sex. https://doi.org/10.1289/EHP1539
  • Received: 23 December 2016
    Revised: 6 September 2017
    Accepted: 18 September 2017
    Published: 31 October 2017

    Address correspondence to J.J. Adibi, 130 Desoto Street, Parran Hall 5132, Pittsburgh, PA 15261 USA. Telephone: 412-624-1913. Email: adibij@pitt.edu

    Supplemental Material is available online (https://doi.org/10.1289/EHP1539).

    The authors declare that they have no actual or potential competing financial interests.

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  • PDF icon Supplemental Material PDF (123 KB)


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    PDF icon Supplemental Table of Contents PDF (114 KB)



Introduction

Phthalates are a class of synthetic, endocrine-disrupting chemicals that are detected in all pregnant women in the United States (Woodruff et al. 2011) Prenatal maternal exposure to phthalates has been associated with short-term outcomes in pregnancy, such as the duration of labor (Adibi et al. 2009; Ferguson et al. 2014; Latini et al. 2003), the risk of preeclampsia (Cantonwine et al. 2016), and fetal sex differentiation as determined at birth (Swan et al. 2015). Long-term outcomes in the children may include negative trends in behavior, IQ, attention, and social communication [11 studies of children 0 to 12 y old reviewed by Ejaredar et al. (2015)].

The role of the placenta in these associations has been partially identified. Placental prostaglandin production, which might be a target of phthalates, is highest at the end of pregnancy and is required for the induction of labor (Tetz et al. 2015). Phthalates may alter circulating levels of antiangiogenic molecules that are produced by the placenta and are indicators of preeclampsia risk (Ferguson et al. 2015). We have identified a potential role of sex-specific placental hormone production in contributing to the effect of phthalate exposures on a neonatal marker predicting the future reproductive health of the child (Adibi et al. 2015a). Clarifying the role of the placenta in these associations will improve temporal and spatial precision in estimating the short- and long-term health consequences of prenatal exposure.

It is not possible to directly observe the critical time points in early pregnancy when mRNA and protein expression in the placenta and in the embryo/fetus are vulnerable to perturbation by tissue and circulating levels of maternal phthalates. Owing to genetic, anatomic, and physiologic differences in placental–fetal biology between species (Maltepe et al. 2010; Rawn and Cross 2008), animal models alone are insufficient to identify causal relationships in human pregnancy. As such, we believe a combination of experimental and observational models that are human-specific and informative of early pregnancy relationships is best suited to deliver these necessary insights.

Another motivation to work simultaneously in experimental and observational systems is the need to overcome a source of intractable confounding: between-person differences in placental metabolism. This is a special problem in phthalate epidemiology because the urinary biomarkers used to assess exposure are also partially products of placental metabolism (Hakkola et al. 1998). At the present time, we have no methods to assess or control for this type of bias. Experimental methods (and statistical methods applied to observational data) may drastically reduce this type of bias and give greater confidence and reproducibility regarding the unconfounded effects of the chemicals on gene and protein expression.

We selected a list of candidate genes to determine if the types of phthalate effects on gene expression that have been identified in other species and in other tissue types were also targets in the human placenta (Adibi et al. 2010). In a companion to this manuscript, we have reported sex-specific associations of eight of these candidate mRNAs measured in term placental tissue biopsies with concentrations of six maternal urinary phthalates (Adibi et al. 2017). Here, we report the experimental follow-up study to those associations using models of undifferentiated and differentiated trophoblasts (Tbs) isolated from first- and second-trimester placentas. In the placental tissue, chorionic gonadotropin α (CGA) was the most strongly associated with the highest number of phthalate metabolites (Adibi et al. 2017). CGA encodes an α subunit of glycoprotein hormones including placenta-specific gonadotropin, called human chorionic gonadotropin (hCG), which is a primary end point in the present study. Here, we follow up on this finding by reporting phthalate effects on mRNAs of both α- and β-subunits of hCG (encoded by CGA and by CGB and its homologues, respectively), on protein levels of intracellular and secreted hCG (dimer of α- and β-subunits), and on the mRNA and intracellular levels of a regulatory factor of hCG, peroxisome proliferation activated receptor γ (PPARG/PPARγ). In this analysis, we compared the molecular response of human Tbs to phthalate exposure according to fetal sex differences, application of single phthalate metabolites versus mixtures, cell type, and consistency between mRNA and protein effects.

Materials and Methods

Study Subjects

Women undergoing elective pregnancy terminations between 7 and 20 wk gestation at the Women’s Options Center at the University of California, San Francisco between 2012 and 2013 donated tissue anonymously. Donation was restricted to women without fetal anomalies. Written informed consents were obtained from all subjects. The University of California, San Francisco Committee on Human Research approved the tissue collection.

Cell Culture

The trophoblast progenitor cell (TBPC) represents an undifferentiated, multipotent population derived from the mesoderm of the chorion at gestational weeks 7.0–14.9. Derivation and maintenance conditions are described elsewhere (Genbacev et al. 2011; Genbacev et al. 2016). TBPCs were cultured in six-well plates that had been incubated with 0.5% gelatin for 30 min. They were plated at 250,000 cells/well. Details on the cell-culture medium are described elsewhere (Genbacev et al. 2016). The villous cytotrophoblasts (vCTBs) were isolated and purified from placentas obtained at 15 wk 4 d to 20 wk 1 d of gestation using microdissection, enzymatic digestion, and cell culture techniques described elsewhere (Hunkapiller and Fisher 2008). Cells were plated on Matrigel® in 24-well plates at 500,000 cells/well, dosed in duplicate, and cultured in serum-free medium (Hunkapiller and Fisher 2008). The vCTBs were sampled from placentas obtained from three female and four male placentas, and the TBPCs were sampled from two female and three male placentas (these cells are referred to hereafter as female and male cells, respectively). Details on experiments and replicates are in the Supplemental Material (see Tables S5 and S6). Single phthalate metabolite doses were designed to mimic maternal urinary concentrations measured in a birth cohort study (Adibi et al. 2017): 200 nM mono-n-butyl phthalate (MnBP; TCI America), 3 μM monobenzyl phthalate (MBzP; TCI), 700 nM mono-2-ethylhexyl phthalate (MEHP; Wako), and 1.5 μM monoethyl phthalate (MEP; Sigma-Aldrich). Specifically, we chose urinary concentrations that in our previous study were associated with CGA mRNA levels and where the association differed in male versus female placentas [Figures 1A, B, D in Adibi et al. (2017)]. In addition, we combined the four abovementioned concentrations of single phthalates into a mixture dose. Vehicle control cells for the single metabolite doses received 0.1% dimethyl sulfoxide (DMSO), and controls for the phthalate mixture received 0.4% DMSO; both of these doses are below the DMSO concentration that affects Tb differentiation (Thirkill and Douglas 1997). The self-renewing TBPC cultures were grown for 72 h, which is the point at which they reach 90% confluency. The vCTB cultures were maintained for 40 h from the time of dosing. vCTBs are nonrenewing; therefore, 40 h is optimal to evaluate functional changes in hCG secretion while cells are metabolically active. In all experiments, cell morphology and cell density were assessed and documented by phase contrast microscopy.

Gene Expression in Cultured Cells

The genes selected for this study were measured previously in placental tissue biopsies and were found to be correlated with maternal urinary phthalates (Adibi et al. 2010, 2017), with the exception of CGB and EREG. CGB was selected because it encodes a β subunit of human chorionic gonadotropin (hCG), a placental hormone that we hypothesize to be a target of phthalate exposure. The CGB primer set amplifies CGB, CGB3, CGB5, CGB7, and CGB8 (TaqMan™, ThermoFisher Scientific). EREG is an ovarian target of luteinizing hormone (LH), a gonadotropin that binds to the same receptor as hCG (Park et al. 2004). In vivo, hCG has been found to stimulate EREG, which is why we also selected EREG for this study (Huber et al. 2007). RNA was isolated using an RNeasy Plus Mini Kit (Qiagen) and measured on a NanoDrop™ spectrophotometer (ThermoFisher Scientific). Reverse transcription was carried out using an iScript™ cDNA Synthesis Kit (BioRad). A TaqMan™ assay for RPS4Y1 was used to assign sex to all control cells. The Ct (amplification cycle at which the mRNA concentration was detectable) for female cells was either >35 cycles, or amplification was nondetectable. For males, amplification of RPS4Y1 was detected at ≤25 cycles. RPS4Y1, a Y-linked gene, was selected for this purpose after reviewing three independently generated transcriptome data sets of placental cells in culture and placental biopsies to determine which of the Y-linked genes showed the greatest discrimination in males and females (J. Adibi, unpublished data, 2013). TaqMan™ gene expression assays (see Table S4) were used for mRNA quantitation, and 10 ng of cDNA was loaded in a 10-μL reaction volume and run in triplicate on a 7900HT quantitative polymerase chain reaction (PCR) instrument (Applied Biosystems). RN18S1 RNA was used as an internal control based on the validated convention of the laboratory where the experiments were designed and conducted (Winn et al. 2007).

hCG in Conditioned Media

Conditioned media from each well from both cell types were aliquoted and stored at −20°C. Aliquots were shipped to the University of Helsinki (Finland), where they were analyzed for a panel of hCG subunits and variants including intact hCG, hCGα, hCGβ, and the hyperglycosylated form of hCG (hCG-h). Intact hCG was measured using time-resolved immunofluorometric assays (IFMAs) (DELFIA®, Perkin-Elmer Wallace), and the other forms were measured using in-house assays with monoclonal antibodies (Alfthan et al. 1992; Lee et al. 2013). The lower limit of quantitation was 0.5 pmol for intact hCG, 5 pmol for hCGα, 1 pmol for hCGβ, and 10.0 pmol for hCG-h. The intact hCG assay measures only the hCG αβ dimer, irrespective of whether it is hyperglycosylated or not. The hCGβ assay measures only free hCGβ subunit, including hyperglycosylated hCGβ. The hCGα assay measures the free α subunit. The hyperglycosylated hCG assay (hCG-h) measures hyperglycosylated intact hCG and hyperglycosylated hCGβ. The hCGα subunit is a gonadotropin subunit that is shared by all glycoprotein hormones, that is to say, hCG, TSH, LH, and FSH. hCGβ lacks hCG activity but may play a role in Tb invasion (Lee et al. 2013).

Quantitative Western Blots

Protein was purified from the same vCTB lysate used for RNA isolation according to the manufacturer’s instructions (AllPrep® RNA/Protein Kit and RNeasy Plus, Qiagen) and was additionally cleaned up by acetone precipitation. Protein lysate was mixed with four volumes of ice-cold acetone and incubated at −20°C for 30 min. After centrifuging at 12,000×g for 10 min, the supernatant was discarded, and the pellet was air-dried. The pellet was then resuspended in 5% sodium dodecyl sulfate (SDS). The protein concentration was measured using a Direct Detect® Spectrometer (EMD Millipore). The protein was denatured at 95°C for 5 min in 4×protein loading buffer (Li-Cor) supplemented with 5% β-mercaptoethanol (Bio-Rad) and separated using Any kD™ Precast Protein Gels (Bio-Rad). The proteins were transferred to nitrocellulose membrane using a Trans-Blot® Turbo™ Transfer System (Bio-Rad). After blocking in phosphate-buffered saline (PBS) blocking buffer (Li-Cor) at room temperature (20–22°C) for 1 h, the membrane was incubated with primary antibodies in blocking buffer at 4°C overnight. The membrane was then washed and incubated with secondary antibodies at room temperature (20–22°C) for 50 min. The reactive proteins were detected using an Odyssey CLx (Li-Cor) imaging system, a method that has been optimized and validated for quantitation (Wang et al. 2007). The Western blot bands were quantified using Image Studio (version 5.0; LI-COR). The primary antibodies were anti-hCG (1:2,000, Dako, A0231), anti-PPAR-γ (1:1,000, Cell Signaling, 24,355), and anti-β-Actin (1:5,000, Santa Cruz Biotechnology, sc4778). The secondary antibodies were IRDye 800CW donkey anti-rabbit IgG (H+L) (Li-Cor) and IRDye 680LT donkey anti-mouse IgG (H+L) (Li-Cor). The hCGβ antibody yielded four bands in the Western blot, of which we quantified three: 42 kDa, 23 kDa, and 21 kDa. The 42-kDa band gave the strongest signal, and the 23- and 21-kDa bands were closest in size to the predicted band. The goal was to determine which band had the highest correlation with CGB mRNA and secreted hCG protein as validation of the Dako antibody and as further insight into possible sex differences in hCG synthetic machinery. The 42-kDa band was 4 log units higher on average than the 23-kDa band and strongly correlated with β-actin in the same sample (r=0.61, p<0.0001). The 21-kDa band was only detected in 57% of female cells and 43% of male cells, and it was not analyzed further (χ2 p=0.02). Results for the 23-kDa band are presented (Figure 3F). The 23-kDa band has been used for hCG quantitation in previous studies (Racca et al. 2011). The PPARγ antibody gave a single band at 51 kDa, consistent with previous studies (Senol-Cosar et al. 2016) (Figure 3G).

Statistical Analysis

For the RNA analysis, mean Ct log2 values (amplification cycle at which the mRNA in the sample was detected; i.e., higher Ct values indicate lower mRNA concentration in the sample) of the technical replicates (n=2) were calculated for each sample. Because of the significant variability in baseline gene and protein expression between biologic replicates and because it is established that hCG also differs significantly by day of gestation (Wald et al. 2003), we chose to analyze the data using multivariate mixed effects models. This method allows us to quantitate differences in gene and protein expression between the treated and control cells after adjustment for the clustering of values within each biologic replicate (random effect) and adjustment for fixed effects (sex, gestational age, etc.). In this case, the random effect or experiment number is a proxy for unmeasured biologic variability at baseline (i.e., genetic and epigenetic variability, maternal health, preprocedure exposures). We included as model covariates gestational age at the time of pregnancy termination, RN18S mRNA levels in the sample, sex of the cells, phthalate dose, and institution at which the experiment was conducted. We report the population marginal means and their 95% confidence intervals (CIs) for all treatment groups by sex. For plotting the changes, we used the β coefficient (equivalent to the ΔΔCt) and its 95% CIs after transformation to the linear scale (2ΔΔct). Sex-specific parameters were calculated, using the Estimate statement in SAS (SAS Institute Inc.), from a model that included a term for phthalate dose by sex. We used two models to estimate all dose effects: the first included the 0.1% DMSO vehicle control samples and the single-metabolite dose groups (also with 0.1% DMSO), and the second included 0.4% DMSO and the phthalate-mixture dose group (also with 0.4% DMSO). Dose group was treated as a categorical variable and interacted on sex.

We applied the same strategy in the analysis of intracellular protein and secreted hCG levels. We report all results for protein on a log scale; a one loge unit difference is equivalent to a 2.7-fold change on a linear scale. For ease of comparison and interpretation, we report log unit differences as fold changes (the difference between the 2 log values transformed to the linear scale). In the analysis of the Western blot data, β-actin was a covariate to control for total protein in the sample. Intracellular hCGβ and PPARγ were modeled as loge-transformed intensity values, and secreted hCG variants were modeled as log-transformed concentrations. To estimate subunit-specific effects, we calculated ratios of the α, β, and hyperglycosylated hCG to intact hCG. This calculation was performed to normalize for overall hCG production, which serves as an indicator of quantity and viability of the cells. We used Spearman rank correlations to quantitate the relationship between mRNAs and intracellular and secreted proteins that were normalized for internal controls. In all mixed effects models, we estimated empirical standard errors that are more robust when the assumption of equal covariance across experiments cannot be confirmed (Verbeke and Molenberghs 2000). Analyses were performed using SAS (version 9.3; SAS Institute Inc.).

Results

The intracellular and secreted hCG protein levels from the TBPCs were primarily below the level of detection and were not analyzed (data not shown). All mRNA and protein end points were well above the detection limit in the vCTBs. Differences in cellular morphology and cell density were not observed in treated versus control cells. We present the sex-specific means (Ct values, protein intensities, and secreted protein concentrations) of cells treated with single metabolites and with the combined metabolites.

mRNA

MnBP stimulated the CGA mRNA expression in female TBPCs (2.1-fold; 95% CI: 1.4, 3.1) and vCTBs (1.3-fold; 95% CI: −0.9, 1.7) (Figures 1A, 2A; see also Tables S1 and S2), whereas an opposite and weaker effect was observed in male cells (TBPCs −0.9-fold; 95% CI: −0.6, 1.5; vCTBs −0.9-fold; 95% CI: −0.7, 1.1). This was the only sex-specific effect on mRNA that was common to both cell types (Figures 1, 2). In the TBPCs, the phthalate mixture increased the CGB levels in female cells 1.4-fold (95% CI: 1.0, 1.9) and decreased the levels in male cells 0.7-fold (95% CI: −0.5, −0.9). In the female cells, the mixture’s effect on CGB resembled the average effect of the single metabolites (1.4-fold vs. 1.5-fold in the mixture-dosed cells). In the male cells, the effect of the mixture was similar to that of MEHP, which significantly reduced CGB (−0.8-fold by MEHP alone vs. −0.7-fold in the mixture-dosed cells). Conversely, in the female vCTBs, the mixture lowered CGB mRNA −0.9-fold (95% CI: −0.8, −1.0) and increased CGB in the male vCTBs 1.7-fold (95% CI: 0.9, 3.2) relative to the controls. There was inadequate biologic replication for the MEP-dosed TBPCs (see Table S5, results not shown).

Figures 1A to 1D are four whisker plots with confidence intervals plotting fold change in TBPC mRNA relative to control cells (mean, 95 percent CI) (y-axis) across phthalates CGA, CGB, and PPARG (x-axis) for metabolites MnBP, MBzP, MEHP, and their mixture.

Figure 1. Phthalate effects on mRNAs. The effects are expressed as mean relative fold change (2ΔΔct) and 95% confidence intervals (CIs) compared with DMSO-treated control cells in undifferentiated trophoblasts (TBPCs). (A) 200 nM MnBP; (B) 3 μM MBzP; (C) 700 nM MEHP; (D) mixture of all four metabolites (MnBP, MBzP, MEHP, MEP). The black lines indicate female-specific effects, and the gray lines indicate male-specific effects. Overall effects that were significant (p≤0.05) are indicated by a line and marked “sex-specific” if the phthalate effect differed in male and female cells. CG, chorionic gonadotropin; DMSO, dimethyl sulfoxide; MBzP, monobenzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MEP, monoethyl phthalate; MnBP, mono-n-butyl phthalate; PPARG, peroxisome proliferator activated receptor gamma.

Figures 2A to 2E are five whisker plots with confidence intervals plotting fold change in vCTB mRNA relative to control cells (mean, 95 percent CI) (y-axis) across phthalates CGA, CGB, and PPARG (x-axis) for metabolites MnBP, MBzP, MEHP, MEP, and their mixture.

Figure 2. Phthalate effects on mRNAs. The effects are expressed as mean relative fold change (2ΔΔct) and 95% confidence intervals (CIs) compared with DMSO-treated control cells in differentiated cytotrophoblasts (vCTBs). (A) 200 nM MnBP; (B) 3 μM MBzP; (C) 700 nM MEHP; (D) 1.5 μM MEP; (E) mixture of all four metabolites. The black lines indicate female-specific effects, and the gray lines indicate male-specific effects. Overall effects that were significant (p≤0.05) are indicated by a line and marked “sex-specific” if the phthalate effect differed in male and female cells. CG, chorionic gonadotropin; DMSO, dimethyl sulfoxide; MBzP, monobenzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MEP, monoethyl phthalate; MnBP, mono-n-butyl phthalate; PPARG, peroxisome proliferator activated receptor gamma.

In the vCTBs, we analyzed three genes that are targets of phthalates in other cell types (Howdeshell et al. 2007; Lovekamp and Davis 2001; Schlezinger et al. 2004) and that are important to placental function: CYP19A1 and CYP11A1 are involved in steroidogenesis and in xenobiotic metabolism, respectively (Hakkola et al. 1998; Miller 1998), and PTGS2 (COX-2) is involved in prostaglandin production by the placenta (Challis et al. 2005). CYP19A1 and CYP11A1 were up-regulated by phthalates in both sexes. MBzP increased CYP11A1 levels in the female cells 1.4-fold (95% CI: 1.2, 1.6) compared with the control cells (see Figure S1). PTGS2 levels were increased in female cells by MBzP 1.2-fold (95% CI: −0.9, 1.6) and decreased in male cells −0.7-fold (95% CI: −0.5, 1.0). In male and female cells combined, MEHP lowered PTGS2 −0.7-fold (95% CI −0.5, −1.0, p=0.02).

Intracellular Proteins

MnBP and MBzP increased hCGβ (2.5-fold and 2.2-fold, respectively) and PPARγ (1.5-fold and 1.8-fold, respectively) (Table 1, Figure 3). The mixture dose had a stronger sex-specific effect on PPARγ than the single metabolites. PPARγ was increased in female cells [1.7-fold (95% CI: 1.1, 2.4)] and was decreased in male vCTBs [−0.6-fold (95% CI: −0.4, 1.1)].

Figures 3A to 3E are five whisker plots with confidence intervals plotting log difference in protein intensity, relative to control cells (mean, 95 percent CI) (y-axis) across phthalates hCG beta and PPAR gamma (x-axis) for metabolites MnBP, MBzP, MEHP, MEP, and their mixture. Figure 3F and 3G are Western blots for hCG beta and PPAR gamma.

Figure 3. Phthalate effects on intracellular hCGβ and PPARγ levels in differentiated cytotrophoblasts (vCTBs). The effects are expressed as the mean natural log difference in protein intensity and 95% confidence intervals (CIs) compared with DMSO-treated control cells. (A) 200 nM MnBP; (B) 3 μM MBzP; (C) 700 nM MEHP; (D) 1.5 μM MEP; (E) mixture of all four metabolites. The black lines indicate female-specific effects, and the gray lines indicate male-specific effects. Overall effects that were significant (p≤0.05) are indicated by a line and marked “sex-specific” if the phthalate effect differed in male and female cells. Examples of (F) hCGβ and (G) PPARγ Western blots. Each dose group was assayed in duplicate (two lanes). This represents a single experiment conducted on cells isolated from a female placenta at 15.6 wk gestation. DMSO, dimethyl sulfoxide; hCGβ, human chorionic gonadotropin β; MBzP, monobenzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MEP, monoethyl phthalate; MnBP, mono-n-butyl phthalate; PPARγ, peroxisome proliferator activated receptor gamma.

Table 1. Mean log intensities of protein (95% CI) measurements of intracellular hCG-β and PPARγ protein expression in vCTBs treated with phthalate metabolites compared with DMSO-treated control cells.
Protein Metabolite Females Males Overall p-Value Sex-specific p-value
hCGβ 0.1% DMSO 3.43 (1.40, 5.47) 4.51 (3.08, 5.94) 3.91 (2.83, 5.43) Reference Reference
hCGβ MnBP 4.63 (4.12, 5.13) 5.00 (3.90, 6.11) 4.81 (4.11, 5.52) 0.08 0.38
hCGβ MBzP 3.94 (2.64, 5.23) 5.43 (4.16, 6.71) 4.68 (3.57, 5.79) 0.04* 0.46
hCGβ MEHP 4.50 (3.84, 5.16) 4.39 (3.14, 5.64) 4.44 (3.57, 5.32) 0.35 0.19
hCGβ MEP 4.18 (2.88, 5.48) 4.72 (3.45, 5.99) 4.44 (3.23, 5.65) 0.35 0.59
hCGβ 0.4% DMSO 4.00 (2.41, 5.60) 4.93 (3.28, 6.57) 4.47 (3.45, 5.48) Reference Reference
hCGβ Mixture 4.41 (3.81, 5.00) 5.21 (3.90, 6.52) 4.81 (4.11, 5.50) 0.34 0.85
PPARγ 0.1% DMSO 5.80 (4.50, 7.10) 5.39 (3.11, 7.68) 5.60 (4.32, 6.87) Reference Reference
PPARγ MnBP 6.05 (4.76, 7.34) 5.99 (4.31, 7.67) 6.02 (4.95, 7.08) 0.01* 0.29
PPARγ MBzP 6.34 (5.57, 7.10) 6.03 (3.95, 8.10) 6.18 (5.12, 7.24) 0.01* 0.81
PPARγ MEHP 6.16 (5.01, 7.31) 5.53 (3.71, 7.34) 5.84 (4.81, 6.87) 0.37 0.65
PPARγ MEP 5.79 (5.14, 6.45) 5.27 (4.15, 6.39) 5.59 (4.83, 6.34) 0.98 0.87
PPARγ 0.4% DMSO 6.36 (5.36, 7.37) 5.94 (4.82, 7.07) 6.11 (5.39, 6.83) Reference Reference
PPARγ Mixture 6.87 (5.95, 7.79) 5.51 (4.67, 6.34) 6.23 (5.45, 7.01) 0.38 0.01*

Note: p-Values are reported for the overall and sex-specific effects of the phthalate dose on secreted hCG. *p≤0.05. Means were estimated by using a mixed effects model with a random intercept for experiment, allowing for control for between- versus within-placenta variability in protein expression. The final sample included three female and three male biologic replicates. Dose groups: MnBP, 200 nM; MBzP, 3 μM; MEHP, 700 nM; MEP, 1.5 μM. The mixture includes all 4 concentrations. CI, confidence interval; DMSO, dimethyl sulfoxide; hCG, human chorionic gonadotropin; MBzP, monobenzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MEP, monoethyl phthalate; MnBP, mono-n-butyl phthalate; PPARγ, peroxisome proliferator activated receptor gamma; vCTB, villous cytotrophoblast cells.

Secreted hCG Isoforms and Subunits

Phthalate effects on secreted hCG levels in the conditioned media differed by metabolite (Table 2, Figure 4). MEP had the strongest effect, with a −0.66-fold decrease (95% CI: −0.59, −0.74) in hCG-h secreted from female cells. In male cells, MEHP increased hCGα secretion 1.21-fold (95% CI: 1.11, 1.33). The mixture significantly suppressed hCGβ (−0.93-fold; 95% CI: −0.88, −0.99) and hCG-h (−0.86-fold 95% CI: −0.75, −0.98) levels in cells of both sexes.

Figures 4A to 4E are five whisker plots with confidence intervals plotting difference in log hCG concentration, relative to control cells (mean difference, 95% confidence intervals) (y-axis) srm of hCG and intact hCG (x-axis) for metabolites MnBP, MBzP, MEHP, MEP, and their mixture.

Figure 4. Phthalate effects on secreted hCG forms in the conditioned media of differentiated trophoblasts (villous cytotrphoblasts, vCTBs). The effects are expressed as the difference in mean natural log concentration and 95% confidence intervals (CIs) compared with DMSO-treated control cells. (A) 200 nM MnBP; (B) 3 μM MBzP; (C) 700 nM MEHP; (D) 1.5 μM MEP; (E) mixture of all four metabolites. The black lines indicate female-specific effects, and the gray lines indicate male-specific effects. Overall effects that were significant (p≤0.05) are indicated by a line and marked “sex-specific” if the phthalate effect differed in male and female cells. DMSO, dimethyl sulfoxide; hCG human chorionic gonadotropin; MBzP, monobenzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MEP, monoethyl phthalate; MnBP, mono-n-butyl phthalate.

Table 2. Mean log concentrations (95% CI) of hCG in the conditioned media of vCTBs treated with four phthalate metabolites, a mixture of the metabolites, and with DMSO.
Protein Metabolite Females Males Overall p-Value Sex-specific p-value
hCGα 0.1% DMSO 7.06 (6.77, 7.35) 6.74 (6.54, 6.94) 6.90 (6.72, 7.07) Reference Reference
hCGα MnBP 7.04 (6.81, 7.28) 6.73 (6.55, 6.91) 6.88 (6.73, 7.04) 0.71 0.95
hCGα MBzP 7.10 (6.75, 7.45) 6.83 (6.65, 7.02) 6.97 (6.77, 7.16) 0.05* 0.49
hCGα MEHP 7.16 (6.83, 7.48) 6.94 (6.72, 7.15) 7.04 (6.84, 7.24) 0.001* 0.18
hCGα MEP 7.05 (6.78, 7.33) 6.84 (6.63, 7.04) 6.94 (6.77, 7.12) 0.29 0.26
hCGα 0.4% DMSO 7.05 (6.73, 7.37) 6.87 (6.59, 7.15) 6.96 (6.76, 7.15) Reference Reference
hCGα Mixture 7.03 (6.64, 7.42) 6.88 (6.57, 7.19) 6.95 (6.73, 7.18) 0.94 0.58
hCGβ 0.1% DMSO 5.14 (4.00, 6.29) 5.62 (4.88, 6.36) 5.38 (4.73, 6.03) Reference Reference
hCGβ MnBP 5.14 (4.10, 6.17) 5.61 (4.82, 6.40) 5.37 (4.73, 6.00) 0.79 0.98
hCGβ MBzP 5.19 (4.10, 6.27) 5.72 (4.96, 6.49) 5.45 (4.82, 6.09) 0.01* 0.26
hCGβ MEHP 5.03 (3.86, 6.21) 5.58 (4.82, 6.34) 5.30 (4.63, 5.97) 0.08 0.33
hCGβ MEP 5.06 (3.90, 6.22) 5.68 (4.88, 6.47) 5.37 (4.69, 6.04) 0.88 0.27
hCGβ 0.4% DMSO 5.19 (3.50, 6.88) 5.94 (4.84, 7.04) 5.56 (4.64, 6.48) Reference Reference
hCGβ Mixture 5.11 (3.44, 6.78) 5.87 (4.77, 6.97) 5.49 (4.57, 6.41) 0.03 0.87
hCG-h 0.1% DMSO 4.47 (3.68, 5.27) 4.65 (4.06, 5.23) 4.55 (4.08, 5.02) Reference Reference
hCG-h MnBP 4.33 (3.44, 5.22) 4.53 (3.91, 5.15) 4.42 (3.91, 4.94) 0.02* 0.79
hCG-h MBzP 4.40 (3.46, 5.33) 4.66 (4.04, 5.28) 4.53 (3.99, 5.07) 0.69 0.37
hCG-h MEHP 4.40 (3.36, 5.44) 4.53 (3.92, 5.15) 4.47 (3.87, 5.06) 0.34 0.78
hCG-h MEP 4.06 (3.19, 4.93) 4.52 (3.92, 5.11) 4.28 (3.78, 4.79) 0.001* 0.01*
hCG-h 0.4% DMSO 4.47 (2.97, 5.98) 4.84 (4.04, 5.64) 4.66 (3.88, 5.44) Reference Reference
hCG-h Mixture 4.36 (2.93, 5.79) 4.69 (3.88, 5.49) 4.52 (3.76, 5.28) 0.01* 0.55
Intact hCG 0.1% DMSO 5.15 (4.48, 5.83) 5.22 (4.76, 5.68) 5.18 (4.78, 5.57) Reference Reference
Intact hCG MnBP 5.05 (4.48, 5.62) 5.16 (4.72, 5.61) 5.10 (4.75, 5.46) 0.21 0.74
Intact hCG MBzP 5.15 (4.44, 5.86) 5.30 (4.87, 5.72) 5.23 (4.83, 5.62) 0.13 0.25
Intact hCG MEHP 5.20 (4.52, 5.87) 5.25 (4.80, 5.70) 5.22 (4.81, 5.63) 0.11 0.66
Intact hCG MEP 4.85 (4.31, 5.38) 5.19 (4.72, 5.67) 5.01 (4.67, 5.36) 0.04* 0.02*
Intact hCG 0.4% DMSO 5.20 (4.27, 6.12) 5.38 (4.78, 5.97) 5.29 (4.77, 5.80) Reference Reference
Intact hCG 0.1% DMSO 5.14 (4.15, 6.13) 5.33 (4.76, 5.91) 5.24 (4.71, 5.76) 0.21 0.79

Note: p-Values are reported for the overall and sex-specific effects of the phthalate dose on secreted hCG. *p≤0.05. Means were estimated by using a mixed effects model with a random intercept for experiment, allowing for control for between- versus within-placenta variability in hCG secretion. The final sample included three female and four male biologic replicates. Dose groups: MnBP, 200 nM; MBzP, 3 μM; MEHP, 700 nM; MEP, 1.5 μM. The mixture includes all 4 concentrations. CI, confidence interval; DMSO, dimethyl sulfoxide; hCG, human chorionic gonadotropin; MBzP, monobenzyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MEP, monoethyl phthalate; MnBP, mono-n-butyl phthalate; PPARγ, peroxisome proliferator activated receptor gamma; vCTB, villous cytotrophoblast cells.

To evaluate specific effects of phthalates on the levels of hCG subunits, we used ratios of the subunits to intact hCG as our end points (see Figure S2). Here, intact hCG serves as an indicator of overall hCG production. We did not detect sex-specific effects. MnBP significantly up-regulated %hCGβ (1.07-fold; 95% CI: 1.00, 1.15), and MEHP significantly down-regulated %hCGβ (−0.89-fold; 95% CI: −0.82, −0.96). MEP up-regulated %hCGα (1.22-fold; 95% CI: 1.06, 1.42) and %hCGβ (1.17-fold; 95% CI: 1.07, 1.28). The mixture did not significantly alter the hCG subunits.

Correlations between mRNA and Intracellular and Secreted Protein

Even though it is only a snapshot, we can evaluate these correlations as indicators of potential sex differences in the underlying in vitro mechanisms of hCG synthesis and secretion. A direct and positive correlation between CGB mRNA and intracellular hCGβ was only observed in females (23 kDa r=0.58, p<0.0001; Figure 5; see also Table S3). This finding is similar to measurements obtained by others who used this antibody, but without consideration of the sex of the placental cells (Uhlén et al. 2015). In males, there was no correlation between levels of mRNAs for hCG and the intracellular protein levels. Correlations that were common to males and females were the negative correlation between CGA and secreted hCGα and the positive correlation between CGB and secreted hCGβ. PPARG mRNA was positively correlated with its encoded protein only in the male (r=0.49, p=0.002) but not the female vCTBs (r=−0.05, p=0.74). These are univariate correlations and do not take into account clustering within placentas or sources of variability in the correlations other than the sex of the cells.

Figures 5A to 5C are flowcharts showing Spearman rank correlations.

Figure 5. Spearman rank correlations between levels of mRNAs, intracellular proteins, and secreted proteins in female and male differentiated trophoblasts (villous cytotrphoblasts, vCTBs). (A) Correlations that are common to male and female vCTBs; (B) Correlations detected only in female and in male cells (p≤0.05). Positive correlations are drawn as solid black lines, and negative correlations are drawn as dotted lines. hCG, human chorionic gonadotropin; PPAR, peroxisome proliferator activated receptor.

Discussion

Using experimental methods, we generated data that reproduced, in cell models, observed relationships between prenatal exposure to MnBP, MBzP, and MEHP and genes essential to placental gonadotropin synthesis (CGA), placental progesterone synthesis (CYP11A1), and placental prostaglandin production (PTGS2). To further evaluate the biological relevance of these changes in mRNA, we measured two corresponding proteins: hCG and PPARγ. MnBP and MBzP changed hCGβ, and MEHP changed hCGα, in ways that were consistent with mRNA effects. To maximize the two-way translational value of our findings to human pregnancy, in the present study, we used primary human placental cells and dosed them with phthalate metabolites at concentrations found in the urine of pregnant women exposed to environmental levels of phthalates. Compared with working with homogenous or immortalized cell lines or high doses of phthalates, this approach presents unique experimental and statistical challenges, yet it produces results with greater translational significance to pregnancy.

In two birth cohort studies, we previously reported that maternal urinary phthalates were associated with higher levels of mRNA and protein in female placentas/fetuses and with lower levels in male placentas/fetuses (Adibi et al. 2015a, 2017). In the present study, we explored this further by studying mRNA and protein effects in tandem and by including a transcription factor that regulates hCG in the placenta that is also activated by phthalates—PPARγ (Fournier et al. 2011; Handschuh et al. 2007; Hurst and Waxman 2003). It has been hypothesized that PPARγ may be the mechanism by which phthalates can exert endocrine-disrupting effects (Desvergne et al. 2009; Lovekamp-Swan and Davis 2003).

Unexpectedly, we observed sex-specific relationships between PPARγ and hCG. PPARG mRNA and PPARγ protein were positively correlated with hCGβ in male but not female cells. hCG synthesis also differed by sex. In female cells, CGB mRNA was positively correlated with intracellular and secreted hCGβ, as expected. In male cells, CGB mRNA was not correlated with intracellular hCGβ. hCGα and hCGβ are subunits of intact hCG but may also have unique functions independent of classical LH/hCG-receptor (Blithe and Nisula 1987; Blithe et al. 1991; Hussa 1980, 1982; Lee et al. 2013). hCGα and hCGβ were highly positively correlated at the mRNA level in both sexes, but not at the protein level. In female cells, the subunits were inversely correlated, and they were not correlated in male cells. hCG subunit variation may be relevant to sex-specific hCG regulation or to other types of posttranscriptional regulation of hCG.

PPARγ may be a key intermediary between phthalate exposure and placental hCG levels, explaining why hormonal effects are opposite in direction for males and females. The effects of the mixture dose on PPARγ were opposite in males and females. The sex difference in the correlation of PPARG with hCGβ and hCGα could explain the opposite effects of phthalates on hCG. These are important and novel insights that give rise to testable hypotheses that can be further studied using biomarkers in human pregnancy and in vitro by using experimental techniques. Additional levels of complexity in this relationship should be considered, such as the epigenetic regulation of PPARG (Lendvai et al. 2016), mitochondrial expression of PPARG in the placenta (Calabuig-Navarro et al. 2016), and sex-specific mitochondrial dysfunction in response to maternal exposures (Muralimanoharan et al. 2015).

We based our experimental doses on phthalate concentrations in maternal urine that were correlated with placental tissue CGA mRNA to compare the two sets of results and to evaluate reproducibility (Adibi et al. 2017). In three cases, the sex-specific associations between phthalates and placental mRNA expression were supported by the in vitro replication. For CYP11A1, the association with MBzP was stronger in magnitude than the in vitro effect. PTGS2 was down-regulated by MEHP in male placentas in both studies. Discrepancy in results between the two study designs may indicate that isolated trophoblast cells, cultured in the absence of fetal tissue and signals from the fetal pituitary/adrenal/gonadal cells, exhibit weaker or even a reversal of hCG sex differences measured in vivo. We observed heterogeneity in the direction and magnitude of the hCG effects by phthalate metabolite, by sex, by hCG subunit, and by differentiated versus undifferentiated Tbs. We offer these as testable hypotheses to be pursued in future studies in vitro and in human populations. hCG is an essential hormone for pregnancy maintenance and is correlated with many obstetric outcomes (Filicori et al. 2005; Yaron et al. 2002b), yet it has not been considered in studies of fetal endocrine disruption to the same degree as androgens, estrogens, and progesterone. In both sexes, there is evidence that hCG can act as a potent gonadotropin at different points in development. In females, hCG is used to stimulate ovulation for the purpose of in vitro fertilization (Yen et al. 2014). In males, hCG has been used to induce virilization and penile growth in prepubertal males with hypogonadotrophic hypogonadism (Bistritzer et al. 1989) and to induce spermatogenesis in adult life. In normal pregnancy, hCG binds to the luteinizing hormone/chorionic gonadotropin (LH/CGR) receptor in the male fetus during the first trimester, stimulating testicular steroidogenesis and thereby indirectly guiding genital differentiation (Huhtaniemi et al. 1977). If the LH/CGR receptor is inactive because of mutation of its gene, males are born with defective genital masculinization (XY, disorder of sexual differentiation) (Kremer et al. 1995). Women with 20% lower circulating hCG had an increased chance of giving birth to cryptorchid boys (Chedane et al. 2014). Taken together, these findings support the idea that disruption of hCG production and function by phthalates or by other endocrine-disrupting chemicals during pregnancy may have effects on fetal sex differentiation.

There are not likely to be real-life situations where a person would only be exposed to one phthalate metabolite at a time; therefore, we evaluated and compared the effects of phthalate mixtures and of single phthalates. We used a nonbalanced approach (i.e., nonequivalent doses) in the design of our doses to accurately reflect real-life exposures during pregnancy (Evans et al. 2012). We detected three cases of a significant effect of the mixture. In the case of CGB mRNA in undifferentiated trophoblasts, the mixture effect was analogous to the single-metabolite effects. We interpret this to mean that there was a common mechanism that was not overwhelmed by the phthalate concentrations used. In the case of intracellular PPARγ in the differentiated trophoblasts, the mixture effect was stronger than the single-metabolite effects, sex-specific, and opposite in direction. The mechanism of PPARγ activation may differ in the case of multiple versus single phthalates. In the case of the female cells, the different metabolites may have synergized to increase the strength of the positive effect on PPARγ (i.e., agonism). In the case of the male cells, the metabolites may have competed for or antagonized (or both) a common mechanism to cause the down-regulation of PPARγ. This latter scenario may also apply to secreted hCGβ, where the negative effect of the mixture was a reversal of the positive effect of MBzP. Comparisons between the effects of single metabolites versus mixtures are critical in establishing which metabolites are more biologically potent and should be prioritized in efforts to reduce risks to the placenta and fetus.

Sex and gestational variation in hCG have been previously established at the population level in analyses of hCG biomarker data (Adibi et al. 2015b; Bremme and Eneroth 1983; Buckberry et al. 2014; Clements et al. 1976; Cowans et al. 2009; Nagy et al. 1994a, 1994b; Steier et al. 1999; Yaron et al. 2002a). This type of variation is generally not considered when analyzing in vitro experimental data. We observed in our primary tissue cultures that the sex and gestational-age variation in mRNA and secreted protein levels that were present at baseline persisted and rendered our biologic replicates less comparable. For this reason, we controlled for these variables in the data analysis using basic multivariate statistical techniques.

There are noteworthy caveats in making comparisons between observational and experimental findings. In the in vitro study, our controls received no phthalates, whereas in the observational studies, we compared pregnancies with lower but not zero exposure because all subjects were environmentally exposed. Similarly, all of the pregnant women were exposed to a phthalate mixture even though we estimated associations with single metabolites to which we compared the results of the present study (Adibi et al. 2017). Phthalate concentrations in placental tissue are most likely lower than the urine concentrations modeled here [phthalates are not measured in blood owing to a short half-life and to a high risk of phthalate contamination in the sampling process (Calafat et al. 2015)]. In a small pilot study, we estimated that MnBP, MBzP, and MEP were higher, but within an order of magnitude, in urine than in placental tissue by 14-, 28-, and 8-fold respectively. MEHP was 4-fold higher in placental tissue; therefore, we may have slightly underestimated the true exposure to the placenta in this study (J. Adibi and N. Snyder, unpublished data, 2017); this may increase the translational value over previously published studies that dosed with concentrations of MEHP that are 1–3 orders of magnitude higher than urinary levels (Meruvu et al. 2016; Tetz et al. 2013; Wang et al. 2016).

These relationships and the relatively small effect sizes are supported by studies conducted by other investigators. In a study of immature rat Leydig cells, CYP11A1 mRNA was 30–40% higher than in controls at 50–500 nM MnBP (Li et al. 2016), similar to the effect we found in male placental cells (33%) in the present study. In an immortalized first-trimester cell line (sex not specified), PTGS, the gene that encodes the COX-2 protein, increased approximately 2- to 3-fold at a 90- μM MEHP dose (129-fold as high as the dose used in our study) (Tetz et al. 2013). In another study, the effects of MEHP on the COX-2 protein were not detected within the dose range that we used, but only at higher doses (Wang et al. 2016). We measured a significant reduction in PTGS2 by MEHP. Results cannot be easily compared owing to the large differences in dose and to a high likelihood of different mechanisms at low versus high doses. In a review of 35 published studies that reported associations of prenatal phthalate exposures with obstetric outcomes, the authors indicated that knowledge of a mechanism and of ways to measure specific biologic intermediaries in human pregnancy are lacking (Marie et al. 2015). Our findings address this gap by offering biologic insight into correlations of prenatal phthalate exposure with placental end points.

Conclusion

In conclusion, we moved one step beyond an observational association by showing sex-specific relationships between MnBP and placental CGA, and between MBzP and placental CYP11A1 using experimental methods with primary cells. The finding that MnBP can alter chorionic gonadotropin α (CGA) was extended to other phthalate metabolites (MBzP, MEHP, and MEP) and to intracellular and secreted hCG and its subunits. The sex-specific effects of phthalates may be challenging to reproduce in vitro in the absence of the fetus. However, our results support the hypothesis that hCG is altered by low concentrations of single and combined phthalates, which is relevant for environmental exposure to phthalates. We are the first to report sex differences in hCG transcription and translation, which we believe to be partially regulated by PPARγ. This finding provides a testable hypothesis to better understand why the hormonal effects of phthalates are opposite in direction between males and females. In future studies, it will be important to quantify the functional significance (for cells, for organs, and for the future child) of small perturbations in hCG by ubiquitous phthalate exposures.

Acknowledgments

We acknowledge S. Fisher and O. Genbacev for their mentoring in placental biology and to Y. Zhou, M. Gormley, N. Hunkapiller, B. Hromatka, and members of the Fisher Lab who all provided valuable mentoring, technical training resources, and feedback on this project. We thank the D. Lewis and S. Gollin laboratories in the Department of Human Genetics and the University of Pittsburgh Genomics Core for support in completing experiments at our new university; we also thank T. Grönholm for technical assistance with hCG immunoassays. Funding was received from the National Institute of Envirnomental Health Sciences/National Institutes of Health (NIEHS/NIH) (grant nos. 1K99 ES017780-01, 5R00ES017780 – 06) and from the Science Innovation Fund of the Passport Foundation (J.J.A.). Funds were provided by the Department of Epidemiology at the University of Pittsburgh to complete this project. Contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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Apple Releases Multiple Security Updates

Original release date: October 31, 2017

Apple has released security updates to address vulnerabilities in multiple products. A remote attacker could exploit some of these vulnerabilities to take control of an affected system.

US-CERT encourages users and administrators to review Apple security pages for the following products and apply the necessary updates:


This product is provided subject to this Notification and this Privacy & Use policy.

WordPress Releases Security Update

Original release date: October 31, 2017

WordPress versions prior to 4.8.3 are affected by a vulnerability. A remote attacker could exploit this vulnerability to obtain sensitive information.

US-CERT encourages users and administrators to review the WordPress Security Release and upgrade to WordPress 4.8.3.


This product is provided subject to this Notification and this Privacy & Use policy.

NTIA Ex Parte Comments on Accelerating Wireline Broadband Deployment by Removing Barriers to Infrastructure Investment

Docket Number: 
WC Docket No. 17-84
Date: 
October 31, 2017
Topics: 

NTIA, as the President’s principal adviser on domestic and international telecommunications policies, and on behalf of federal agency purchasers of telecommunications services, respectfully comments on the Notice of Proposed Rulemaking in this proceeding. NTIA supports the Commission’s proposals for streamlining the section 214 process by which Title II-regulated carriers may discontinue service. We suggest ways to ensure that the discontinuance and copper retirement processes can accommodate the needs of federal communications users.

20170820 – Mechanistic modeling of developmental defects through computational embryology (WC10th)

Abstract:
An important consideration for 3Rs is to identify developmental hazards utilizing mechanism-based in vitro assays (e.g., ToxCast) and in silico predictive models. Steady progress has been made with agent-based models that recapitulate morphogenetic drivers for angiogenesis, somitogenesis, urethrogenesis, and palatogenesis. Additional models are underway for the neurovascular unit, endocardial cushions, and neural tube closure. The models offer a heuristic approach to reconstruct tissue dynamics from the bottom-up, cell-by-cell and interaction-by-interaction. Individually, they can simulate emergent phenotypes and predict adverse outcomes or ‘cybermorphs’ to essentially bring an AOP to life utilizing computational dynamics. Collectively, their compilation into an integrated array or ‘virtual embryo’ motivates the construction of novel ontology systems to integrate molecular pathways, cellular behaviors, and in vitro data on chemical-biological interactions with extant knowledge of embryology. This abstract does not reflect US EPA policy.

20170910 – Computational Systems Toxicology: recapitulating the logistical dynamics of cellular response networks in virtual tissue models (Eurotox_2017)

Translating in vitro data and biological information into a predictive model for human toxicity poses a significant challenge. This is especially true for complex adaptive systems such as the embryo where cellular dynamics are precisely orchestrated in space and time. Computer cell agent-based models (ABMs) that incorporate the logistical dynamics of complex signaling networks built in CompuCell3D can be wired to recapitulate key morphogenetic events. An array of embryologically-inspired ABMs or &lsquo;virtual embryo&rsquo; provides an approach to in silico generation of developmental phenotypes or &lsquo;cybermorphs&rsquo; by electronically manipulating the underlying biological network. By imputing toxicity profiles from in vitro assays on key genes, pathways or cellular behaviors, a series of concentration-response curves may be translated into predicted adverse outcomes for developmental toxicity. This provides a novel approach to translate concentration-response profiles from high-throughput screening (HTS) libraries such as ToxCast/Tox21 into a probabilistic prediction of developmental toxicity. Combinations can be tested in silico for cumulative or aggregate exposures as well as chemical-interactions with nonchemical stressors. Model outputs to date include quantitative predictions of effects on VEGF-mediated angiogenesis (angiodysplasia), androgen-mediated urethral closure (hypospadias), and TGF&szlig;-mediated tissue fusion (cleft palate). Other virtual tissue models underway include the limb-bud (phocomelia), endocardial cushion (valvulo-septal defects), and neurovascular unit (microcephaly). Disclaimer: This abstract does not reflect EPA policy.

Evidence of Standing Waves in Arc Jet Nozzle Flow

Abstract: Waves spawned by the nozzle in the NASA Ames 60 MW Interaction Heating Facility arc jet were experimentally observed in pressure surveys at the exit of the nozzle. The waves have been seen in past CFD simulations, but were away from the region where models were tested (for the existing nozzles). However, a recent test series with a new nozzle extension (229 mm exit diameter) revealed that these waves intersect the centerline of the jet in a region where it is desirable to put test articles, a…

Comparison of indoor air sampling and dust collection methods for fungal exposure assessment using quantitative PCR

Evaluating fungal contamination indoors is complicated because of the many different sampling methods utilized. In this study, fungal contamination was evaluated using five sampling methods and four matrices for results. The five sampling methods were a 48 hour indoor air sample collected with a Button™ inhalable aerosol sampler and four types of dust samples: a vacuumed floor dust sample, newly settled dust collected for four weeks onto two types of electrostatic dust cloths (EDCs) in trays, and a wipe sample of dust from above floor surfaces. The samples were obtained in the bedrooms of asthmatic children (n = 14). Quantitative polymerase chain reaction (qPCR) was used to analyze the dust and air samples for the 36 fungal species that make up the Environmental Relative Moldiness Index (ERMI). The results from the samples were compared by four matrices: total concentration of fungal cells, concentration of fungal species associated with indoor environments, concentration of fungal species associated with outdoor environments, and ERMI values (or ERMI-like values for air samples). The ERMI values for the dust samples and the ERMI-like values for the 48 hour air samples were not significantly different. The total cell concentrations of the 36 species obtained with the four dust collection methods correlated significantly (r = 0.64–0.79, p < 0.05), with the exception of the vacuumed floor dust and newly settled dust. In addition, fungal cell concentrations of indoor associated species correlated well between all four dust sampling methods (r = 0.68–0.86, p < 0.01). No correlation was found between the fungal concentrations in the air and dust samples primarily because of differences in concentrations of Cladosporium cladosporioides Type 1 and Epicoccum nigrum. A representative type of dust sample and a 48 hour air sample might both provide useful information about fungal exposures.

Comparison of Multiscale Method of Cells-Based Models for Predicting Elastic Properties of Filament Wound C/C-SiC

Abstract: Three different multiscale models, based on the method of cells (generalized and high fidelity) micromechanics models were developed and used to predict the elastic properties of C/C-SiC composites. In particular, the following multiscale modeling strategies were employed: Concurrent multiscale modeling of all phases using the generalized method of cells, synergistic (two-way coupling in space) multiscale modeling with the generalized method of cells, and hierarchical (one-way coupling in spa…

A Generic Software Architecture For Prognostics

Abstract: Prognostics is a systems engineering discipline focused on predicting end-of-life of components and systems. As a relatively new and emerging technology, there are few fielded implementations of prognostics, due in part to practitioners perceiving a large hurdle in developing the models, algorithms, architecture, and integration pieces. As a result, no open software frameworks for applying prognostics currently exist. This paper introduces the Generic Software Architecture for Prognostics (GS…