Document aB1jmDEoz9mbbDp013bkoOMre

P-1 Taft/ T a ft S te ttin iu s & H o lliste r LLP 4 2 5 W a ln u t S tree t, S uite 1 8 0 0 '' C in c in n a ti, OH 4 5 2 0 2 -3 9 5 7 / Tel. 5 1 3 .3 8 1 .2 8 3 8 / Fax; 5 1 3 .3 8 1 .0 2 0 5 / w w w .ta ftla w .c o m C incinnati / Cleveland / C olum bus / D ayton / Indianapolis / N orthern Kentucky / Phoenix / Beijing Ro be r t A. B ilott 513-357-9638 bilott@ taftlaw.com July 7, 2010 EPA Docket Center, MC 2822T U.S. Environmental Protection Agency EPA West, Room 3334 1200 Pennsylvania Avenue, NW Washington, D.C. 20460-0001 Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQQRD-2003-0016 To IRIS Database for PFOA/PFOS: In response to the Notice issued by USEPA on February 23, 2006, regarding USEPA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk Information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23, 2006), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR-226 database: 1. New Castle, Delaware, Municipal Services Commission Customer Flyer on PFOA/PFOS Test Results (2010). RAB:mdm Enclosure cc: Gloria Post (NJDEP)(w/ end.) (via U.S. Mail) Helen Goeden (MDH)(w/ end.) (via U.S. Mail) Lora Werner (ATSDR)(w/ end.) (via U.S. Mail) 11850104.1 P-2 We Thought You Should Know If you-hay?-seen the latest edition o f National Geographic magazine the whole issue is devoted to the subject o f water. Just as people are concerned about Global Warming there are those who are trying to raise the public's awareness o f the world's water supply and the threats it is under. Regulated and Unregulated Sources of Contamination Every Juaethe MSC publishes it's Consumer Confidence Report and mails it to every residence in the City. The CCR discloses to the public all EPA (Environmental Protection Agency) regulated substances found hi your drinking water and their Maximum Contaminate Leyels (MCLs). Currently there is no regulated substance which exceeds the Maximum ; m your drinking water. The regulated substances are both naturally occurring in the environment and those that are man-made. Teflon Precursors (PFO A and PFOS) A customer requested the MSC test for the active chemicals found in Teflon (PFOA and PFOS). This citizen was concerned about newspaper reports that trace amounts were showing up in the drinking water supplies in New Jersey and Maryland. He felt there was a reasonable . expectation sve may also be exposed since we share the same aquifer. The Commissioners agreed that the MSC should test for the presence even though the EPA does not regulate these two chemicals. Test Results Two sets o f test were done and both showed the presence o f PFOA and PFOS, the active ingredients in Teflon in the MSCs source water. The MSC shared this information with the Office o f Drinking Water and the State Toxicologist. Since these are not regulated substances the EPA has not set an MCL for its presence. The State suggested that we take samples at the tap to measure its presence in our treated water. The MSC will do this but it felt it should notify its customers about its presence. ; Parts Per Billion -f-i Teflon precursors are measured in parts per billion. One (I) part per billion is theequivalent to a teaspoon of water in an Olympic size pool (660,250 gallons) or a single penny in - ' $10,000,000. The MSC found at its highest concentration that PFOA was 1/T6 o f a singlepenn) and PFOS was equal to one penny. Conclusion The MSC will do further testing and keep its customers advised about any risk they may be exposed to. S p rin g lif ta E ll flushing will stasi ssiiiaf, Upgil 12th P-3 "McCrea, Deborah" <mccrea@taftlaw.com> 07/02/2010 04:07 PM To NCIC OPPT@EPA cc "Bilott, Robert A." <bilott@taftlaw.com>i ~ bcc Subject 07/02/2010 Letter To EPA Docket Center Taft / Celebrating 125 Years Deborah McCrea / Legal Assistant Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800 Cincinnati, Ohio 45202-3957 Tel: 513.381.2838 Fax: 513.381.0205 www.taftlaw.com / mccrea@taftlaw.com Internal Revenue Service Circular 230 Disclosure: As provided for in Treasury regulations, advice (if any) relating to federal taxes that is contained in this communication (including attachments) is not intended or written to be used, and cannot be used, for the purpose of (1) avoiding penalties under the Internal Revenue Code or (2) promoting, marketing or recommending to another party any transaction or matter addressed herein. This message may contain information that is attorney-client privileged, attorney work product or otherwise confidential. If you are not an intended recipient, use and disclosure of this message are prohibited. If you received this transmission in error, please notify the sender by reply e-mail and delete the message and any attachments. p.4 Taft/ T a ft S te ttin ius & H o llister LLP 425 W a ln u t Street, Suite 1 8 0 0 /C in c in n a ti, OH 4 5 2 0 2 -3 9 S 7 /T e l: 513.381 2 8 3 8 /F a x : 513.381.0205 / w w w .ta ftla w .c o m C incinnati / Cleveland / C olum bus / D ayton / Indianapolis / N orthern Kentucky / Phoenix / Beijing Ro b er t A. B ilott 513- 357-9638 bilott@taftlaw.com July 2, 2010 EPA Docket Center, MC 2822T U.S. Environmental Protection Agency EPA West, Room 3334 1200 Pennsylvania Avenue, NW Washington, D.C. 20460-0001 ~j Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQORD-2003-0016 To IRIS Database for PFOA/PFOS: In response to the Notice issued by USEPA on February 23, 2006, regarding USEPA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23, 2006), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR-226 database: 1. Genuis, S.J., et al., "Human Detoxification of Perfluorinated Compounds," Public Health (doi:10.1016/j.puhe.2010.03.002) (online June 2010); and 2. Guerrero-Preston, R., et al., "Global DNA Hypomethylation is Associated With In Utero Exposure to Cotinine and Perfluorinated Alkyl Compounds," 5:6 Epigenetics 1-8 (August 16, 2010). RAB:mdm Enclosures cc: Gloria Post (NJDEP)(w/ end.) (via U.S. Mail) Helen Goeden (MDH)(w/ end.) (via U.S. Mail) Lora Werner (ATSDR)(w/ end.) (via U.S. Mail) Reginald Jordan (NC SAB) (w/encl.) (via U.S. Mail) 11844530.1 P-5 1 P-6 PUBLIC HEALTH XXX ( 2 0 1 0 ) I - g available at w w w .sciencedirect.com Public Health jo u rn a l hom epage: w w w .elsevierhealth.com /journals/pubh Review Paper Human detoxification of perfluorinated compounds SJ. G en u is"'", D. Birfeholz0, M. R alitsch55, N. Thibaultb 3University of Alberta, Canada bA.L.S. Laboratory Group ARTICLE INFO Article history: Received 24 August 2009 Received in revised form 10 February 2010 Accepted 2 March 2010 Available online xxx Keywords' Bile acid squestrants Cholestyramine Detoxification Enterohepatic circulation Perfluorinated compounds Public health Saponin compounds Sauna therapy Toxicology Zeolites SUMMARY There has been no proven method thus far to accelerate the clearance of potentially toxic perfluorinated compounds (PFCs) in humans. PFCs are a family of commonly used synthetic compounds with many applications, including repelling oil and stains on furniture, clothing, carpets and food packaging, as well as in the manufacturingofpolytetrafluoroethylene-a non stick surfacing often used in cookware (e.g. Teflon(r)). Some PFCs remain persistent within the environment due to their inherent chemical stability, and are very slowly eliminated from the human body due, in part, to enterohepatic recirculation. Exposure to PFCs is widespread and some subpopulations, living in proximity to or working in fluorochemical manufacturing plants, are highly contaminated. PFCbioaccumulation has become an increasingpublic health concern as emerging evidence suggests reproductive toxicity, neurotoxicity and hepatotoxicity, and some PFCs are considered to be likely human carcinogens Acase history is presented where an individual with high concentrations of PFCs in serum provided: (1) sweat samples after use of a sauna; and (2) stool samples before and after oral administration of each of two bile acid sequestrants - cholestyramine (CSM) and saponin compounds (SPCs). Stool samples before and after use of a cation-exchange zeolite compound were also examined. PFCs found in serum were not detected in substantial quantities in sweat or in stool prior to treatment. Minimal amounts ofperfluorooctanoic acid,but no other PFCs, were detected in stool after SPC use; minimal amounts of perfluorooctanesulfonate, but no other PFCs, were detected in stool after zeolite use. All PFCcongeners found in serum were detected in stool after CSMuse. Serum levels of all PFCs subsequently declined after regular use of CSM. Further study is required but this report suggests that CSM therapy may facilitate gastrointestinal elimination of some PFCs from the human body. 2010 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved. Introduction Over the last two decades, there has been increasing discussion in the scientific and public health literature about adverse effects of bioaccumulative toxicant exposure.1,2 A family of chemical agents w hich has garnered increasing attention in toxicological and environm ental literature is the perfluorinated compounds (PFCs), both parent compounds and their metabolites. Concern has arisen because the ultimate breakdown products of per fluorinated parent chemicals are perfluorinated acids (PFAs), some of which are persistent in the environment and can accu m ulate in the hum an body and in food chains. Emerging evidence in animal research and in preliminary hum an epidemiology studies suggests the potential for toxicity with ' Corresponding author. 2935-66 Street, Edmonton, Alberta T6K 4C1, Canada. Tel : t-1 780 450 3504; fax: +1 780 490 1803. E-mail address: sgenuis@ualberta.ca (S.J. Cenuis). 0033-3506/$ - see front m atter 2010 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.puhe.2010.03.002 Genuis SJ; e t a!., H um an detoxification o f perfluorinated com pounds. Public Health (201); p.7 2 PUBLIC HEALTH XXX (3 0 I 0 ) ! - 9 exposure and accrual of PFAs. The recently released `Fourth National Report on Human Exposure to Environmental Chem icals' from the Centers for Disease Control and Prevention confirms th at most Americans have bioaccumulated perfluorochemicals in their bodies.1In this paper, a briefoverview of the current understanding of the significance of PFC toxicant exposure is followed by a discussion of PFC elimination and a case report involving hum an PFC excretion after therapeutic intervention. Perfluorinated compounds PFCs are a family of m an-m ade com pounds w hich have been m anufactured over the last 60 years and have been used in various commercial applications. Structurally, these chem ical agents consist of a linear or branched carbon backbone th at is entirely substituted by strong bonds to fluorine atoms. The fluorine com ponent of PFCs provides extremely low surface tension and accounts for their unique hydrophobic (water repelling) and lipophobic (lipid repelling) nature.3 These compounds differ markedly from m ost other chem ical surfactants in that they are very stable, non-reactive and effective at low concentrations. With such unique properties, selected PFCs have been used to m ake com m ercial products th a t are re sista n t to both w ater and oil, and can also w ith stand the extrem es of tem perature, pH and oxidizing conditions. PFCs have primarily been used in domestic and industrial contexts specifically for their repellant properties. With the propensity to deter stains and prevent adhesion, these compounds have been utilized in clothing, cookware, furniture, carpeting and with various other applications to prevent stains and sticking. Animal studies suggest that PFCs are generally well absorbed by oral intake and inhalation. As a result, in domestic and commercial settings, many people have regular exposure to PFCs through inhalation, ingestion and, perhaps, dermal contact. In addition, accidental spills of PFCs have recently occurred w here individuals have been exposed to elevated levels of these agents through avenues such as the water supply.4With increasing presence of these agents in the environment and bioaccumulation in some individuals, research to understand PFC toxicokinetics and modes of action is currently underway.4,5 Toxicokinetics PFCs appear to be primarily excreted by the liver and possibly the kidney. Although they often enter the intestine as a compo nent of bile, it appears th a t m ost PFCs may be re-absorbed and returned to the liver through th e enterohepatic circulation3 where the cycle of biliary excretion and re-absorption recom mences repeatedly.8 In addition, there is increasing evidence in the literature that persistence in the body may also result from impaired renal excretion of some PFCs due to renal tubular re absorption mediated through organic anion transporters.9"12 Although there has been discussion of the im pact o f pH changes on renal re-absorption of some toxicants, determinants of renal excretion are not completely understood and there is no known clinical intervention at this juncture to preclude PFC re absorption in the kidney. In review, certain precursor PFCs can be m etabolized in the body to PFAs, these acids are not further metabolized and are eliminated at a slow rate, they tend to accrue in the organism, and they distribute mainly to th e serum , liver and kidney,13 with hepatic levels reaching values much higher than serum concentrations in som e anim als.14,15 It is uncertain, however, w hat th e relative concentrations for various PFAs are betw een blood and various tissues in hum ans as there appears to be variance of tissue distribution between species and between different PFA com pounds.13,16 After PFC parent com pounds are metabolized to PFAs, however, it appears th at these persistent compounds in serum are highly bound to protein an d are repeatedly shunted to th e liver and kidney en route to attem pted clearance and subsequent re-absorption, resulting in prolonged circulating levels of some PFAs after exposure has occurred. Individual chain-length homologues within families of PFCs have a considerable range of elimination rates and half-lives but, as discussed, some PFAs tend to be very persistent in the hum an body. The median hum an serum half-lives for common PFAs are: PFHxS, 8.8 years (range 2.8-27); PFOS, 5.4 years (range 2.4-21.7); and PFOA, 3.8 years (range 1.5-9.1).17 Between indi viduals, there may be variation in biological breakdown of precursors and elimination o f PFAs due to differences in indi vidual biochemistry and physiology based on genomic and metabolic variation. A gender difference in elimination of PFCs in hum ans has not been observed thus far.7 Due to the bio accumulative nature o f some PFCs, there is increasing concern about adverse health sequelae; an apprehension which has recently spawned much international attention and increasing research. There is significant chemical variation within the family of PFCs that accounts for marked differences in behaviour and potential toxicity betw een specific perfluorinated molecules. Some compounds such as perfluorobutyrate, for example, appear to be readily excreted with a low potential for bioaccumulation.6 Other PFCs, however, are persistent, non-biodegradable and bioaccumulative within th e hum an body.1,3'' The three most widely know n PFCs - perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS) and perfluorohexansulfonate (PFHxS) - have significant potential for endogenous accrual after exposure. Although initially thought to be biologically inactive, the persistence of these agents within the hum an organism has generated increasing concern about th e potential for some PFCs to disrupt physiological processes. Potential toxicity Most PFC toxicity work thus far has been done on anim als. In anim al research, common PFCs such as PFOA and PFOS appear to be potentially carcinogenic,18induce functional alteration in cellular organelles,19"21 and cause neurotoxicity and hepatotoxicity.18,22,23 The literature also confirms significant PFC adverse effects on im m une system function,24 cell m em brane potential,25 neuroendocrine function,26 and gestational and developm ental processes.27,28 In sum mary, anim al research evidence to date confirms th at som e PFCs have potential for hepatotoxicity, developmental toxicity, immunotoxicity, hormonal disruption, and genomic and biochemical impact.3 Although the aforementioned effects often occur at higher inieas ctt^ .th^ i^ de .doi:100l6/j.puh2010.C3.00 Hman deto^catidn-of.perfluoriritd compounds,' PubUc Healthst2010),;: .' : p.8 PUBLIC HEALTH XXX (2 0 I 0 ) 1-9 3 doses th a n those w hich m ost h um ans are exposed to. potential hum an thresholds for harm are currently unknown. With increasing recognition of verified toxicity in short-term animal studies, research on the short- and long-term im pact of PFAs on hum an health has increased. In-vitro studies on cells derived from hum ans have demon strated th a t som e PFCs may be genotoxic,29some have impact on intracellular organelles,30 and som e dem onstrate oestrogenic effects.31 In population studies, there is recent evidence that hum an fecundity is affected by som e PFCs, with the suggestion that som e of these compounds may act as horm one disrupters.12 In addition, a large Danish cohort study recendy dem onstrated that fetal birth weight is impaired by background exposure to PFOA.33 Although findings are not yet conclusive, the Science Advisory Board of the Environmental Protection Agency from the US G overnm ent has found th a t there is sufficient evidence to consider PFOA to be a likely carcinogen in hum ans.34 In addition, there has been vigilant observation of a large pop ulation of people in W est Virginia (approximately 100,000) who sustained substantial exposure and bioaccumulation of PFCs d u e to drinking w ater contam ination a t an industrial site. Prelim inary findings in studies on exposed individuals suggest a num ber of adverse sequelae including altered hepatic function, im m une function, thyroid function and cholesterol physiology.5 Therapeutic interventions There is very limited inform ation in the literature about interventions to facilitate elim ination of PFCs.3SOne report in the scientific literature assessed the use of a bile acid s q u estran t (BAS) cholestyram ine (CSM) to facilitate excretion of som e PFCs in rats: CSM enhanced PFOS and PFOA excretion by approxim ately 9.5-10 fold.36 The researchers also found th a t levels of retained PFCs in liver and plasm a after CSM treatm ent were significantly diminished as a result of enh an ced excretion.36 It has also been noted in som e prelim inary unpublished work that intervention with non absorbable fats may h asten th e rem oval of PFCs in animals. Jandacek assessed th e 48-h excretion of PFOA in anim als 7 days after dosing w ith Olestra (an agent acting as a lipophilic sink to im pair fat absorption); excretion of PFOA was doubled in th e treated group.32 R esearchers at 3M, a m ajor producer o f PFCs, claim to have investigated th e effectiveness of colesevelam HCL (an o th er BAS noted for its preferred tolerability) in lowering blood levels of PFOA in cynom olgus m onkeys.38 Under conditions of a preliminary study, they report that colese velam HCL did no t appear to be highly effective in dim in ishing seru m PFOA values, b u t investigations for PFOS or PFHxS elim ination were n o t perform ed. As sulfonate com pound s m ay bind m ore strongly th a n PFOA, it is unclear if colesevelam HCL m ight have a m ore pronounced effect w ith PFOS and PFHxS. To summarize, there is minim al to no research to be found on successful therapeutic interventions to remove persistent PFCs from th e h u m an body. This paper is the first to report on therapeutic m easures to facilitate hum an excretion of PFCs in an individual noted to have high levels of PFCs in serum testing. Case history Patient description A 51-year-old asym ptom atic male medical researcher, who volunteered to be a control participant in an institutional review board (Health Ethics and Research Board a t the University of Alberta) approved research study on toxicant bioaccumulation and excretion in chronically ill patients, w as unexpectedly found to have markedly elevated serum levels of selected PFCs. A ssorted other chemicals w ere explored in this study including blood, urine and sw eat levels of polybrom inated diphenyl ethers, phthalates, bisphenol A, solvents, heavy metals, organochlorine pesticides and poly chlorinated biphenyls. No elevated levels of toxicants other than serum PFCs were found in the blood, sw eat or urine of th e volunteer (hereinafter referred to as the `p atient'). Upon receiving the results, th e patient requested th a t all family m em bers who had lived in th e sam e residence be tested for PFCs. Approval by the University of Alberta Health Ethics and Research Board was also received for a subsequent pilot study to test and provide ongoing follow-up for the patient and his family, and to assess the potential source of the exposure by retrieving various samples from within their household. All other individuals living in the sam e household w ere eventually found to have a profile of elevated PFCs, including one child with a serum PFHxS level of 423 ng/g and a PFOS level of 108 ng/g [the 95th percentile from the NHANES study (2003/2004) was 8.3 for PFHxS and 54.6 for PFOS39). Information gathered through personal interviews revealed that the family lived in Edmonton, Alberta, Canada a locale w ith latitude of SS'SCN and a climate of long, cold winters. Their place of residence was a house heated by in floor heating, made up of water-filled rubber tubing running immediately beneath the sub-floor. The in-floor heating was turned off in the sum m er m onths but was used routinely between September and May each year. On inspection, their hom e was found to have poor ventilation and air exchange. Most of the house, including all the bedrooms, was floored with carpeting, which had been treated interm ittently for stain and soil repellence since 1989. No apparent occupational exposure or unusual dietary habits were discovered for the patient or any members of the household. Samples of dust and carpet collected from the family's hom e eventually confirmed high levels o f PFCs, particularly PFHxS. Within his clinical medical practice, the first author has repeatedly found markedly elevated PFC levels (usually PFHxS and PFOS) in serum sam ples of residents (unrelated to the patient in this case report) living in carpeted hom es with in floor heating, but not in residents of carpeted hom es w ithout in-floor heating systems (including homes with carpets receiving interm ittent Scotchgard treatments). The reasons for elevated PFC exposure in this patient are the subject of ongoing investigation, but the preliminary hypothesis (which received additional support from the findings of noteworthy PFC levels in the carpets and air in all the carpeted room s within his home) was that off-gassing from heated carpets w hich had been interm ittently treated w ith Scotchgard (tm) for carpet stain resistance and protection, released PFCs into P-9 4 PUBLIC HEALTH XXX (2010) t -9 the air w hich were inhaled by the patient and members of his family. The carpets were completely removed from the home in January 2009. There was discussion about the health history ofthe family, as the patient's spouse had sustained two consecutive mid-term pregnancy losses shortly after moving into this home, whereas her previous obstetrical and health history had been unremark able. With concern about the well-being of his family and wishing to preclude potential health problems associated with elevated PFCs, the patient researched the scientific literature extensively with regards to the potential sequelae associated with PFC bio accumulation. As independent emerging evidence found in the scientific literature demonstrated potential adverse biological effects,32,33,40 the participant was eager to consider options to diminish PFC levels with the objective of minimizing associated health risks. Over many months, he sought advice and counsel from individuals involved in environmental health sciences and toxicological research on possible interventions in order to reduce the levels ofPFCs for him and his family. He also contacted a major manufacturer of PFCs and, contrary to information from other sources, was adamantly told that there were absolutely no risks associated with elevated PFC concentrations in people, in food webs or in the environment, and was also informed that therewas no known intervention to hasten hum an excretion of PFCs. The patient explored and accum ulated information relating to assorted possible therapies that might facilitate and hasten excretion of bioaccumulative compounds, including use of BASs,41-44 pancreatic lipase inhibitors,8 n o n absorbable lipids,44-47 sauna depuration,48-50 serial phle botomy (as is used in haemochromatosis) and therapeutic apheresis. He also sought advice about assorted complemen tary and alternative therapies, including the use of herbal agents such a s zeolites51"53 and saponin com pounds (SPCs). SPCs-originating from soy or the yucca plant are alleged to have a BAS m echanism of action.54-57 After consultation with various researchers including toxicologists and environmental health scientists, the patient chose to try the BAS CSM for various reasons: ease of use, ready availability of medication, and absence of invasive intervention; long-term CSM has been employed for other indications (including treatm en t o f elevated lipids) and h a s an excellent track record of safety; CSM is no t absorbed into th e body, b u t binds strongly to selected adverse compounds and is excreted along with the toxicant. W ith lack of absorption, CSM is considered by som e clinicians to be less potentially toxic th an m any overthe-counter medicaments in common usage; anim al w ork h as confirmed th a t CSM m ay facilitate excre tion o f som e PFCs36; the side-effect profile of CSM is very tolerable (although long term use of CSM may cause a deficiency of fat-soluble nutri ents including vitamins A, D, E and K, and coenzyme Q10, nutrient supplementation ingested a few hours away from medication intake can be used to m aintain nutrient levels); and repeated a n d long-term adm inistration of CSM (more th an 3 years) has reportedly been used by some clinicians for m ould- an d mycotoxin-exposed ill p atients w ith good results and w ithout noticeable adverse effects.58-50 It is im portant to note th a t th e self-directed treatm ent plan using CSM by th e individual in this case history w as under taken of his own volition. As well as deciding to em bark on a trial of treatm ent with CSM. the patient decided to explore the potential efficacy of sauna depuration as a m easure to facilitate elimination of his accum ulated PFCs. To determ ine th e potential for success, he initially decided to use CSM for 1 week and to have post treatm en t stool PFC levels measured. At a later point, he repeated the sam e procedure w ith SPCs and zeolites to determine if these non-pharmaceutical agents might also facilitate PFC elimination. The PFC results from each of the family m em bers and th e household contents will be discussed in subsequent papers after adequate analysis is completed; this paper provides a case history to report the results of selfdirected CSM use in this individual patient. Sample collection Prior to com m encing CSM treatm ent, the patient collected blood, sweat, urine and stool sam ples for PFC assessm ent. Three independent blood draw s were taken in the year prior to com m encing treatm en t in order to confirm and follow levels of PFCs. Recognizing th a t induced perspiration can be an effective m eans to excrete som e chemical toxicants,18-50 sweat was collected during three sessions in an infra-red dry sauna. The patient showered prior to using the sauna and brushed off the area of skin from the trunk and axillae area w here perspiration was to be collected. A stainless steel spatula was used to collect the sweat, and the body fluid was placed into a glass jar and delivered to the laboratory for assessm ent. The urine and faecal samples were collected directly into glass collection jars provided by the laboratory. In June 2008, the patient com m enced CSM (4 g, three times per day, for 1week); stool testing was performed on the 1-week post-treatm ent sam ples. Levels o f PFCs in serum , urine, sw eat and stool prior to and after treatm en t with CSM can be found in Table 1. After results were reviewed, it was felt th a t the PFC levels from stool sam ples post-CSM intake provided justification for a trial of ongoing treatm ent with CSM. Continuing after the 1 week trial, the patient continued to take CSM (4 g, three times per day) for approximately 20 consecutive weeks in order to diminish his body burden of PFCs. In December 2008, the patient decided to try SPCs (from Yucca Schidigera Saponin: `Optimum D-Tox' brand; 8 drops, twice a day, for 1 week) and repeated the stool sample in the same fashion. The carpets were removed from the house on 20 January 2009, and subsequent testing confirmed that the target PFCs found in the individual and family members were isolated from the air samples and carpet in their home. After reading in the scientific literature that another class of agents called zeolites has the ability to facilitate removal of selected toxicant compounds by cation exchange,51,61,62the patient also elected in November 2009 to try a zeolite compound (zeolite-containing clinoptilolite: 'Waiora' brand; 3 drops, three times a day, for 1 week), and repeated the stool sample before and after using the zeolite compound. No other medications were being used at the tim e w hen SPCs or zeolites were used. The patient and his family members intend to pursue therapeutic intervention to eliminate accrued PFCs, doi-101016/ji'p u h e2 0 1 % 3 fc^ 2 ; ' Si. gt-aL, -Human detoxificatioiiof p^uorinated cbmppUnds,' PutjliqiHealth (2010)," 5 T i;- ;'- - ' V" ' `< p. 10 PUBLIC HEALTH XXX ( j O l o ) l - g 5 \% l | * a 1I 85888888 |W ~ 5 5" 8. ? S& S 1 %S S'S 1 -<S S* :Sg -JET.-'88 W8i :cStj. '-OECL".OS,u<<uvU 88838888 S :.. g ^ <ri oS' * <3/> E01'Sv> gR4>/. 2MfOtlJ mC S-:g<9 Ot.'S as [`:.o l*JJ^ 8 8 8 S 8 8 8 8 t _ g .cj j ; _Q rV* TV^ TVH H VH TVHVrirVi i*il* !W ^ <U OO "CO 41 5 oo Hrt S S 5* w S |g g j;O. ! ' ?* ! lA^OOOOOO M'S s s s M^ .4^ <e :s u c u) I ' f e ' i . u . CO :% Z g ! J. Is i s ^ t 1;W 2 ;w Nq mc j i n i f l wi r vS o"> ov o o ov ov dy 3E |3*j -82S.:f1' CoS" u "g ^ | 8f 61 z I S.S; a n J -g /Pi ' . Q S g ? :|-a 8 J.- S '& <N & lls .Xo.p OM vOt vOi /0t i0n i AOiOn uoi ov dv dv vo vo vd vd -vd g *> C 1s1 | I 8- I 5 S g.l. -a 8 kf -.w: N* 4 * i0n i0n 0, lo 0ts>0 0 la 0vS T-* dV oV OV dVoV' dV oV ga uu. -e5c8? '?I5s 1*|*.33 2 o *o irt _P fet n rv g f I % ;:CijiJ-i;cicSm. 'IS'^SbOJ oO-oonddodi0n 0in 0in Os i oin oin i0n 0tft y y v v v .v v 3 - O 1 p5Vo8Cfi:'?-&<Pu.-.'lSS2Sav".-.3gaeMfi!` 3 u I t? E `S '." mm* S&ikh'.AUL -S - . 01 u : ,c > --r-iUdC4?/>:J>-^Cto>f-. d'*o 6 ;1 ^ xo ^ r-2 -;^u2 1-g 15?:'^' I*' w* 1 OIHX. CN ...S-.-o&: |'i ^ 8 mm hlefat5-.. m id; o .vc o SdV\vdVo:^wovd. '-3v o' 'f e :>: ,< v o ; 'xua- .-o0Om.^p&-<-.0jao .:'Czpt Udt Ud*V;qE - au-.&i\-0+ -cUum'C^5. and to be followed to assess PFC levels in the m onths and years to come. PFC analysis Formic acid {0.1 M) and a m ixture of isotopically labelled PFCs (Wellington Laboratories, Guelph, Ontario, Canada) were added to 1.0 ml of serum , urine or sweat. The m ixture was vortexed, sonicated and subjected to solid-phase extraction. The extract was concentrated to 100 |ri and an instrum ent perform ance internal standard was added along w ith 200 nl of 90% 20 mM acetic acid/10% methanol. For faecal PFC analysis, m ethanol along with a m ixture of isotopically labelled PFCs w as added to 1.0 g of freeze-dried stool. The m ixture was vortexed, sonicated and centrifuged. A known am ount of the extract w as collected and concentrated to 100 |d on a nitrogen evaporator; 200 jil of 90% 20 mM acetic acid/10% m ethanol and instrum ent performance internal standard were added to the vial. Analysis for each sam ple was perform ed by liquid chro matograph tandem mass spectrometry using multiple reac tion monitoring. Detection limits were 0.5 ng/ml for each analyte, based on the lowest standard in the standard curve. Procedural blanks were run with each set of samples, and blanks w ere always below 0.5 ng/ml. As m entioned, the extracts were concentrated to 100 jil. To secure precision, the laboratory staff added 100 nl of methanol and carefully marked the meniscus. When the sample was concentrated, it was brought to the meniscus line previously marked in order to keep the volumes reproducible. The isotopically labelled standards used were: (i) 13C4-PFOS, sodium perfluoro-l-[l,2,3,4-l3C4|octanesulfonate; (ii) 13C4-PFOA, perfluoro-n-[l,23,4-u C4]octanoic acid; (iii) UCS-PFNA, perfuloro-n'[l,2,3,4,5-13C5)nonanoic acid; and (iv) n C2-PFDA, perfluoro-n-[l,2-i:!C2]decanoic acid. All of the standard calibra tion curves were linear. The standard concentrations used were: (i) 0.5 ppb; (ii) 2.0 ppb; (iii) 10.0 ppb; (iv) 30.0 ppb; and (v) 50.0 ppb. Results and discussion The PFC results for serum, urine, sweat and stool sam ples are provided in Tables 1 and 2. The results of this case study suggest th a t PFOA was the only PFC readily excreted in urine prior to any therapeutic intervention. Only miniscule am ounts of PFHxS were detected in the sw eat sample; no other PFCs were found in sweat, thus PFCs did not appear to be excreted readily into perspiration after sauna therapy and this was not investigated further. Following SPC treatm ent, only miniscule am ounts of PFOA were detected in the faecal sample. No other PFCs were detected, thus SPCs did not appear to facilitate considerable excretion of PFCs into stool. Following zeolite treatm ent, only minuscule am ounts of PFOS were detected in the faecal sample; no other PFCs were detected, thus the type of zeolite used in this case did not appear to facilitate considerable excretion of PFCs into stool. Prior to treatm ent by CSM, no PFCs were detected in stool. Following CSM treatm ent, however, PFHxS, PFOS, PFOA and PFNA were identified in both post-treatm ent faecal samples. Serum levels repeated after 12 and after 20 weeks of CSM treatm ent dem onstrated an apparent decline in PFHxS, PFOS, /RV - 'V PUBLIC HEALTH XXX (2 0 1 0 ) 1-9 Table 2 ~ Serum levels of perfluorinated com pounds prior to and after treatm ent w ith cholestyram ine (CSM). 1 Analyte : Srum (ng/g) June 2007 Prior to treatm ent Serum (ng'g) March 2008 Prior to treatm ent 'Serum (ng/g) June 2008 Immediately prior to treatm ent Serum'(ng/g) September 2008 After approx. 12 weeks of CSM treatm ent Serum (ng/g) November 2008 After approx. 20 weeks of CSM treatm ent Carpets removed from home in January 2009 PFHxS ' PFOS PFOA PFNA 60.0 26.0 6.8 - 0.9 59.1 58.0 ' 27.4 ' 23.0 ; S.5 ' 5:9 0.7 :. o.s 50.4 15.6 4.4 . <0,5 46.8 14.4 4.1 <0.5 - - ' PFHxS, peril-uorohexansulfonate; PFOS, perfluorooctanesulfonate; PFA, perfluorooctanoic acid; pfna, perfluorononanoic acid. PFOA and PFNA levels {Table 2). Use of CSM in this patient, therefore, appears to be associated with and may facilitate excretion of som e bioaccumulated PFCs, m ost notably PFHxS, PFOS a n d (perhaps) PFOA. Stool sam ples taken from another family m em ber using CSM for elevated serum PFCs also showed significant excretion o f these compounds into feces. As PFCs are surfactants like endogenous bile acids, it is not surprising th a t the CSM acts as a resin and interrupts recycling of these compounds. Further study is required, however, to determ ine the generalized applicability of these preliminary findings to other PFC contam inated individuals. With emerging evidence about the potential health sequelae associated with hum an PFC accrual juxtaposed with (i) wide spread exposure to bioaccumulative PFCs in the general pop ulation, and (ii) select subgroups with sizeable exposures to PFCs,4 it behooves the medical and public health comm unity to delineate potential interventions which diminish the accumu lated load of these persistent toxicants. Although conclusive generalized information cannot be gleaned from an individual case history, the findings in this case report suggest that selected bile acid sequestrants may have therapeutic value in the elimi nation o f certain persistent PFCswithin the hum anbody. In order to verify this, a carefully designed prospective research study involving several PFC-contaminated participants is required. In review, the question arises as to w hether the CSM treat m ent translated into significant reductions in the body burden of PFCs. AccordingtoTable2, three consecutive values spanning the y ear from June 2007 to June 2008 showed no substantial decline in serum levels of PFHxS and PFOS. However, w ithin 3 m onths o f BAS use, the levels of all elevated PFCs declined steadily and consistently, despite ongoing exposure (as the carpets w ere not yet removed). Accordingly, the treatm ent may have been responsible for the reduction in serum levels. The most compelling finding in this case, however, is that faecal levels of PFCs were present w ith use of CSM. Concluding thoughts There is increasing global attention to the problem of persis tent pollutants and their sequelae, both in the environment and w ith in the hum an body.1,63 Emerging research suggests that accrued toxicants may lead to illness and increased health risk throughout th e life cycle.3,63' 65 W ith the 21st century reality of escalating toxicant exposure and bioaccumulation am ong individuals and population groups,1 research is now underway to find potential interventions to facilitate excretion of persistent toxicants in order to diminish the body burden and to preclude or overcome associated health problems (See Box l).66 Furthermore, som e health providers and researchers involved in molecular medicine and environmental health sciences are recommending clin ical and laboratory assessm ent for individuals with possible toxicant bioaccumulation,67' 69 and are exploring and employing interventions to preclude and address such bio accum ulation w hen possible.41' 434i'49'66'7<>`79 As PFC hum an health research is a relatively new under taking, it rem ains to be seen to w hat extent these compounds contribute to adverse health outcomes and hum an suffering. While there is increasing evidence suggesting health sequelae associated with PFC bioaccumulation, there are some authors and publications that have minimized concern about the accrual of these chemical agents. It is imperative to recognize th a t much of the research related to potentially toxic compounds, including PFCs, is undertaken or funded by the specific companies, industry-supported organizations and affiliated scientists involved with the production of the chem icals being studied. Furthermore, some toxicology journals routinely use reviewers from chemical companies to scrutinize and review manuscript submissions about the compounds their company manufactures. With increasing attention to conflict of interest concerns,80-84 the ongoing debate about how to approach the issue of patients' health versus company profits,81 and increasing awareness of num erous egregious violations by some scientists and lobbyists working to conceal industry culpability,80 it is som etimes diffi cult to discern the credibility of research which has received commercial funding from companies producing potentially toxic agents. It is also problematic when credible papers high lighting potential sequelae of toxicant exposures are dismissed because of commercial influence in the journal review process. Therefore, it is recommended that regulatory intervention be enacted whereby: (i) scientific journals require th at research be registered and funded through arms-iength independent research bodies where researchers, study design, reporting and publication of evidence are not in a conflict position, and are not acquiescent to the sway of comm ercial power and influence; and (ii) scientists working for industry be excluded from the peer review process of papers related to their company's products. p. 12 PUBLIC HEALTH XXX ( 20 1 0 ) 1-9 7 In the literature, there are no reported studies of successful interventions to remove PFCs from the hum an body. Exposure to these commonly used non-stick and stain-resistant com pounds is widespread, b u t excretion from the body is im paired as a result of the chemical nature of some of these agents and their propensity to be re-absorbed in the kidney and the enterohepatic circulation. With emerging evidence about th e possible toxicity associated w ith accrual of PFCs, in ter ventions to hasten rem oval in individuals with high levels may be indicated. This paper presents a case history suggesting that treatm en t with CSM may facilitate elim ination o f commonly encountered PFCs, including PFOA, PFOS and PFHxS. Sauna depuration, treatm ent w ith SPCs and u se o f zeolites do not appear to be effective m eans of facilitating the excretion of PFCs. Other interventions, such as therapeutic apheresis, which have the ability to remove protein-bound toxicants w arrant investigation as potentially rapid methods of elimi nating PFCs.66Further study is required, b u t the use of selected BASs such as CSM shows prom ise as one type of possible intervention for facilitating th e elim ination of PFCs in hum ans. : Box 1!Public health recom m endations regarding PFCs 1) Individuals'w ho reside or have previously lived in ,.-:: . c a p ^ t e d dwellings withjm-floor-.or baseboard heating sh o u ld be-tested for.bioaccum ulated PFCsi 2) fu r th e r research should b e undertaken to confirm th a t so m e .BASs such a s CSM effectively facilitate the excretion o f certain PFCs. 3) '-O th e r em erging ,interventions, such as therapeutic . apheresis, which have the ability to remove proteinv. b o u n d toxfcanjs .w arrant investigation a s potentially rap id m ethods o f elim inating PFCs. 4) ."Iiidividuals> with significantly elevated levels of PFCs shouljibe.ajpprisedbffhe potential risks associated with - u,I5fe>bioaccumulation, an d should be `offered the opportunity-toreceive therapy to facilitate elimination . v.of fhese.com pounds,in order,to dintinishjthe risk of adverse health outcomes. 5) .Research o n th e h ealth im pact of- potentially toxic . - chem icals should be registered a n d funded through arrnsr-length independent research bodies w here researchers involved in study design, reporting and . /publication of/evidence are not in a conflict position -* -//and areio t-in flu eticed by-com m ercial'interests. exclude scientists hirediby L ^ ^ ^ ^ ^ ^ i ^ d u s t r y - , f t p m .the /peer/-review applications, reseamh|proposp}s_,and ^ ^ d 't ip ^ 'm a n u fa c r .. 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Treatment with a dietary fat substitute decreased Arochlor 1254 contamination in an obese diabetic male. J Nutr Biochem 2005;16:383-4. 76. Rea WJ, Pan Y, Johnson AR, Ross GH, Suyama H, Fenyves EJ. Reduction of chemical sensitivity by means of heat depuration, physical therapy and nutritional supplementation in a controlled environment. J Nutr Environ Med 1996;7:141-8. 77. Aposhian HV, Maiorino RM, Gonzalez-Ramirez D, ZunigaCharles M, Xu Z, Hurlbut KM, et al. Mobilization of heavy metals by newer, therapeutically useful chelating agents Toxicology 1995;97:23-38. 78. Imamura M, Tiing TC. A trial of fasting cure for PCB-poisoned patients in Taiwan. Am; Ind Med 1984;5:147-53. 79. Roehm DC. Effects of a program of sauna baths and metavitamins on adipose DDE and PCBs and on clearing of symptoms of agnet orange (Dioxin) toxicity. Clin Res 1983; 31:243. 80. Michaels D. Doubt is their product: how industry's assault on science threatens you health. New York: Oxford University Press; 2008. 81. Olivieri NE. Patients' health or company profits? The commercialisation of academic research. Sci Eng Ethics 2003;9: 29-41. 82. Angell M. Is academic medicine for sale? N Engl ; Med 2000; 342:1516-8. 83. Angell M. The truth about the drug companies: how they deceiue us and what to do about it. New York: Random House; 2004. 84. Topol EJ. Failing the public health - rofecoxib, Merck, and the FDA. N Engl J Med 2004;351:1707-9. &rrian'dta^caii6h'ofT^Muoflntdvc'dtopotmd^:Pubhc;FIealth (2010); p. 15 2 RESEARCH PAPER Global DNA hypomethylation is associated with in utero exposure to cotinine and perfluorinated alkyl compounds Rafael Guerrero-Preston,1-2* Lynn R. Goldman, 2 Priscilla Brebi-Mieville,14 Carmen Ili-Gangas,14 Cynthia LeBron,' Mireya Hernndez-Arroyo,5 Frank R. Witter,6 Ben i. Apelberg,7 Marina Roystacher,18 Andrew Jaffe,7 Rolf U. Halden'-9 and David Sidransky' 'Otolaryngology Department; a n d ` Department of Environmental Health Sciences, an d` Department of Gynecology and Obstetrics. School of Medicine; and 'Department o f Epidemiology, Bloomberg School of Public Health; The Johns Hopkins University; Baltimore, MD USA; ^Universidad de Puerto Rico; Escuela Graduada de Salud Pbflca; Departamento de Salud Ambiental; Ro Piedras, Puerto Rico;'Universidad de La Frontera; Facultad de Medicina; Oepartamentode Anatoma Patolgica; Laboratorio de Patologa Molecular; Temuco Chile: ' Institute of Forensic Science of Puerto Rico; San Juan, Puerto R ic a ` Hadassah University; Hospital Mount Scopus; Department of Surgery; Jerusa lem, Israel; ` Arizona State Uni versity; The Btodesign Institute, Tern pe, A Z USA Ke y words; epigenomics, umbilical cord serum, hypomethylation, cigarette smoke, perfluoroocrane sulfonate, perfluorooccanoate, global D NA mthylation Abbreviations: BMI, body mass index; PFCs, perfluorinated alkyl compounds; PFOA. perfluorooccanoate; PFOS, perfluorooctane sulfonate; THREF,, cracking health related to environmental exposures Environmental exposures in utero may alter the epigenome, thus impacting chromosomal stability and gene expression. We hypothesized that in utero exposures to maternal smoking and perfluoroalky! compounds (PFCs) are associated with global DNA hypomethylation in umbilical cord serum. Our objective was to determine if global DNA m thylation could be used as a biomarker o f in utero exposures to maternal smoking and PFCs. Using an ELISA-based method, global DNA mthylation was quantified in umbilical cord serum from 30 newborns w ith high (>10 ng/ml, mean 123.8 ng/ml), low (range 1--10 ng/ml, mean 1.6 ng/ml) and very low (<1 ng/ml, mean 0.06 ng/ml) cord serum cotinine levels. Ychromosome analysis was performed to rule out maternal DNA cross-contamination. Cord serum global DNA mthylation showed an inverse dose response to serum cotinine levels (p < 0.001). Global DNA mthylation levels in cord blood were the lowest among newborns w ith smoking mothers (mean = 15.04%; 95% Cl, 8.4, 21.7) when compared to babies of mothers who were second-hand smokers (21.1%; 95% Cl, 16.6, 25.5) and non-smokers (mean = 29.2%; 95% Cl, 20.1, 38.1). Global DNA mthylation was inversely correlated with serum PFOA (r = -0.72, p < 0.01) but not PFOS levels. Serum Y chromosome analyses did not detect maternal DNA cross-contamination. This study supports the use o f global DNA mthylation status as a biomarker o f in utero exposure to cigarette smoke and PFCs. Introduction Epigenomics provides a window through which we can under stand the impact of che environment, nutrition and lifestyle choices on chronic disease susceptibility and risk.1 Life-long effects of in utero exposures may be mediated through alterations in the best-understood epigenomic mark, DNA methylation. Global D N A methylation, one of the earliest molecular changes observed in the transition from a normal to a diseased cell, may be a good epigenomic bio-dosimeter of prenatal exposures to environmental toxicants that lead to impaired health through out the life-course.2Global DNA hypomethylation largely affects intergenic and intronic regions, particularly repeat sequences and transposable elements, and is believed to result in chromosomal instability and increased mutation events. Environmental exposures have been shown to be associated with epigenetic changes. Arsenic, one o f the heavy metals found in cigarette smoke, has been shown to be associated to global DNA hypomethylation in vitro' and in animal models'1and to be associated with in utero D NA hypomethylation.'' Cadmium, a metal associated with smoking and second hand smoke expo sure has been associated with DNA hypomethylation along with induction of cell proliferation.'' Cadmium accumulates in the placenta, reduces birthweight and may also play a role in uterine growth restriction.7 Polycyclic aromatic hydrocarbons (PAH), also components of cigarette smoke, have been associated with ` Correspondence to: Rafael Guerrero-Preston; Email: rguerre3@jhmi.ed j Submitted: 02/28/10; Accepted: 05/14/10 Previously published online: www .landesbioscience.Lom /journals/epigenetics/article/12378 DOl: I0.416l/epi.5.612378 www.landesbioscience.com Eplgenetics 1 Table 1. Characteristics o f infants selected fo r epigenetics study com pared w ith ail eligible infants. Baltimore THREE study Epigenetics study Eligible births (not included) om il e n (%) n =83 n(%) p-value* Male infant IS (50%) 47 (57%) 0.49 African American 27 (90%) 61 (74%) 0.54 M other <18 yr 4 (13%) 7 (9%) 0.45 M other >30 yr 7 (23%) 23 (28%) 0.80 Baltimore residence 25 (83%) 60(72%) 0.22 Parity: firs t birth 11 (37%) 48 (58%) 0.65 Prenatal care first 4 months 23 (77%) 72 (88%) 0.15 Smoking by history 8 (27%) 9(11%) 0.04 Mean (SD) Mean (SD) p-value" Serum cotinine (ng/ml) Very Low (VL) (<1 ng/ml) 0.06 (0.04) 0.16 (0.22) 0.20 Low(L) (1-TO ng/ml) 2.0(1.3) 2.5 (2.1) 0.59 High (H) (>10 ng/ml) 135.6(94.3) 121.8(87.7) 0.79 levels o f DNA mthylation in mononuclear cells from whole blood o f adult women. Brecon et al." reported decreased global DNA methylacion in buccal cells from kindergarcen and first grade children who were exposed to maternal smoking in utero. However, these studies evaluated DNA methylacion in different tissues, and both studies are limited by having used surrogate measurements o f global D N A mthylation, having determined in utero smoking exposure retrospectively'by questionnaires, and not having controlled for exposure to second hand smoke over time.12 At this time chere are no published studies that examine the association between global D N A mthylation at birth and in utero exposures to smoking or to persistent environmental contaminants. To determine if global DNA methylacion in newborns is asso ciated with exposure to prenatal maternal smoking we measured global DNA methylacion in cord blood serum o f full term babies with adequate birchweighc, and high, low and very low cocinine levels in cord serum. As a secondary analysis we also examined the association between levels of DNA methylacion and persis tent perfluoroalkyl compounds (PFCs) in cord serum: perfiuorooccane sulfonate {PFOS) and perfluomoctano3te (PFOA). Serum cotinine (ng/ml) by smoking history No 11.8(34.2) 0.16 (0.23) 0.004 Yes 140.1 (106.4) 68.5' (88.5) 0.15 Serum cotinine (ng/ml) by smoking history and cotinine levels Nonsmokers (No & cot-VL) Passive smokers (No & cot-L) 0.06 (0.02) 2.0 0.2) 0.16(0.03) t 0.20 N/A Smokers (Yes & /or cot-H) 135.6' (94.3) 68.5' (8B.5) 0.13 PFOA (ng/m l) 1.8 (0.89) 1.8 0.09) 0.84 PFOS (ng/m l) 5.8(3.12) 6.1 (3.55) 0.72 Birth w e ig h t (g) 3408 (378) 3336 (506) 0.48 Length (cm) 50.7 (1.8) 50.4 (2.5) 0.52 Ponderal index 2.61 (0.043) 2.60 (0.033) 0.88 Head circumference (cm) 33.9 (1.3) 33.9(1.6) 0.84 Gestational age (days) 278.3 (6.9! 274.8 (7.6) 0.03 Paternal age (years) 27.4 (7.4) 28.0(7.6) 0.74 Maternal prepregnancy BMI 27.8 (8.0) 26.2 (6.1) 0.30 DNA m ethylation (%S-mC) 21.0 00.3) - N/A 'Chi-square. " t-test. 'All eligible passive smokers were included in this study. T w o w ith no smoking history had cotinines in the active smoking range, 117and 118 ng/mL respectively and are classified as smokers. 'One w ith a smoking history had cotinine below the active smoking range, 4 n g /m l. altered global D N A mechylarion in peripheral blood leucocytes o f male nonsmoking coke-oven workers.11 A handful of studies have examined the association between tobacco smoke exposure and global D NA methylacion. Active smoking is known to be associated with global DNA hypomechylation in the aero-digestive mucosa.1' In addition, two studies have reported inconsistent results documenting associations between prenatal tobacco smoke exposure and global DNA methylacion. Terry et al."1 reported that prenatal exposure resulted in higher Results Among the 30 babies included in this study, 13 were male and 15 were females. As shown in Table 1, those included in this study were similar to all eligible babies except that there was by design oversampling o f babies whose mothers were active and passive smokers, a tendency for the mothers in this sub study to be somewhat less educated and a trend coward these mothers hav ing been iess likely ro have received prenatal care in the firsr four months of pregnancy. As expected, nonsmokers in the sample had somewhat lower levels o f serum cocinine than in all eligible babies. Maternal age ranged from 6 co 37 years and paternal age ranged from 15 to 49 vears. Seven mothers were <18 years o f age and seven were 30 or older. One mother was Asian, two were Caucasian, and the other 27 were African American. O f the 11 smokers chat were selected, two had reported that they did nor smoke but were classified as smokers on rfie basis o f having very high serum cotinine levels (117 and 118 ng/ml). Means, medians and ranges of D N A mthylation index results (%5 mC), PFOS and PFOA levels, gestational age and growth parameters also are presented in Table 1. The subjects selected for global mthyla tion analysis were similar in ail ways to the rest o f the eligible infants with the exception o f gestational age, which was on aver age 3.5 days longer. Table 2 shows the relationships between D NA mthyla tion index results and selected maternal and infant characteris tics. Maternal age did not show a linear relationship to DNA mthylation. However, both younger (<20 y) and older (30+ y) mothers had babies with reduced global DNA mthylation (NS). Likewise there was a non-significant trend toward increased D NA mthylation with increased gestational age within this group of term births (p = 0.19). There was no evidence chat DNA mthylation was associated with pre-pregnancy BMI, or sex of the baby. Measures o f infant growth-- birchweighc, length, head 2 Epigenetics Volume 5 Issue 6 Table 2. Results o f linear models (pearson's correlations and slope beta values) for cotinine, PFOA, and PFOS concentrations (log-transformed) and o th e r selected study variables on %5-mC (n = 30), baltim ore three study Crude Model 2* Model 3 r beta p-value beta p-value beta p-value Cotinine (ng/ml) -0.39 -0.048 0.04 -0.054 0.05 -0.043 0.06 In (Cotinine) -0.62 -1.99 <0.001 -2.11 <0.001 -1.91 0.001 Smoking status None (comparison) Passive Active -8.07 0.051 -9.48 0.03 -8.17 0.06 * -14.5 0.002 -15.3 0.001 -13.7 0.004 PFOA (ng/ml) -0.32 -3.74 0.08 -3.39 o.n -3.20 0.15 Ln (PFOA) -0.35 -7.59 0.06 -7.00 0.07 -6.61 0.11 PFOS (ng/ml) -0.21 -0.71 0.26 -0.367 0.58 -0.52 0.42 Ln (PFOS) -0.28 -5.36 0.13 -3.45 0.37 -4.46 0.22 Maternal age (yrs) <20 * -5.96 0.18 20-29 30+ (comparison) * -7.60 0.12 Maternal age (yrs) -0.138 -0.236 0.46 Gestational age (days) Sex (male) 0.245 * 0.366 0.16 0.19 0.97 Prepregnancy BMI 0.016 0.022 0.93 Birthweight (100 gm) -0.17 -0.46 0.37 Length (cm) -0.18 -1.03 0.33 Head circumference (cm) 0.02 0.88 0.56 Ponderal index 0.03 1.28 *N/A. 'Adjusted for maternal age. "A djusted for gestational age. 0.88 circumference and ponderal index-- also were not associated with global D NA methylation. Table 2 also shows results o f multivariace linear regression models for global D N A methylation in relation to exposures to cotinine levels, smoking status and levels o f PFOS and PFOA. Cotinine, the natural log of cotinine, active smoking and passive smoking were significantly associated with global D NA hypomethylation. The associations for PFOA and the natural log of PFOA were of borderline statistical significance. PFOS was not associated with changes in global DNA methylation. Multiple regression models were done first including maternal age (in cat egories) and rhen gestational age as covariaces. Inclusion o f either maternal age or gestacional age somewhat attenuated the effect estimates across the board. However, in both sets o f multivariate models the associations between cotinine and smoking status and DNA methylation were statistically significant. Figure 1 presents the DNA mechyiation data by smoking sta tus. The global methylation index was significantly (p < 0.01) lower for 11 newborns exposed to maternal smoking (mean = 15.04%; 95% Cl, 8.4, 21.7) than among 8 newborns with non smoking mothers (mean = 29.2%; 95% Cl, 20.1, 38.1). The serum D NA global methylation index also was significantly (p = 0.01) lower for 11 newborns whose morhers were exposed to secondhand smoke during pregnancy (mean = 21.1%; 95% Cl, 16.6, 25.5) when compared to newborns not exposed to maternal smoking. Figure 2 presents the results o f linear regression modeling o f D N A methylation index resulcs across the three groups, in www.landesbiosdence.com Epigenetics 3 a different systemic compartment, Additionally, our secondary analyses raise che possibility that DNA global hypomethylation in cord serum is associated with pre-natal exposures to perfluoro- alkyi compounds, especially PFOA. Cord blood irrigates al! the cells of the fetus collecting cell free DNA that can be isolated from 8 cord serum. Therefore, DNA extracted from cord serum provides us a genomic footprint o f feral cellular turnover. However, since there have been sporadic reports o f maternal DNA in cord Sp o o. blood11 we tested the precedence of che DNA we extracted from cord blood. Serum Y chromosome analyses did not detect maternal DNA cross con tamination in cord blood. Therefore, we are mea suring global methylation levels in fetal DNA isolated from cord serum. Non-smoker Passive Smoker Active Smoker Global DNA methylation levels have been found to be inversely associated with blood Figure 1. Global DNA mthylation index in cord blood serum DNA o f 11 newborns exposed in utero to tobacco smoke constituents from maternal smoking, 11 newborns whose mothers w ere passive smokers, and 8 newborns whose mothers were non-smok ers. (Box plots). plasma levels for several persiscent organic pollut ants (POP) in Greenlandic Inuit2' and in cissues from animals exposed to ionizing radiation.2' Low-dose exposure to POPs has recently been associated with global DNA hypomethylation ina healthy adult population.25 POPs such as organo- relation to log-cransformed cocinine, PFOA and PFOS levels. chlorine (O C) pesticides, which are xenobiotics that accumulate There was a statistically significant inverse relationship between in adipose tissue, may be chronically exerting their deleterious the natural log o f cocinine measured in cord blood serum and che effect on the epigenomic machinery. These recent findings have global DNA methylarion index (Fig. 2A and for PFOA (Fig. 2B) profound implications for future generations also, since chemical- but not PFOS (Fig. 2C)). Serum PFOS and PFOA levels were induced epigenetic changes are heritable through generations.26 not related to maternal smoking stacus or to serum cocinine levels In addition, in utero exposures to toxic compounds may lead to (data not shown). deleterious effects on the epigenomic molecular machinery and To ensure that we were measuring global D N A methylation subsequent adverse reproductive outcomes. changes in fetal DNA and not in maternal DNA, we quanti It has been documented that in utero exposure to dioxins, fied che Y chromosome content in umbilical cord serum DNA PCBs, O C pesticidesor bisphenol-A may cause adverse pregnancy for a subset of 8 male infants. One sample was noc quantifiable ouccomes such as low birth weight, preterm delivery, or intrauter leaving 7 for statistical analysis. The ratios o f Y Human DNA ine growth retardation.2''28 For example, there is already evidence to tocal Human DNA (mean = 6.7) would reflecc the amount that in utero exposure to bisphenol-A leads to D NA hypomethy o f male infants' DNA relative to the amount o f maternal DNA lation of HoxalO, with a subsequent alteration of the develop that might be present in cord blood o f the same patient. Any mental programming o f uterine estrogen response in an animal ratio above one is indicative that the sample is from a male infant model.29 Global DNA hypomethylation is commonly found in without the presence of female DNA. Results range from 1.6 to most types o f cancer. Higher degrees o f genome-wide hypo 15.5, so that there is no measurable level of maternal DNA in methylation have been found to be strongly associated with late cord blood serum, implying that the observed global methylation grades and with larger tumor sizes, suggesting that genome-wide changes are o f feral and not maternal origin (Table 3). de-methylacion occurs simultaneously with tumor progression.10 Emerging evidence indicates that such alterations also occur in Discussion ocher chronic diseases and may be associated to environmental exposures in utero." Our results suggest that circulating fetal DNA already can Dietary-induced epigenecic changes are also heritable through exhibit aberrant methylation patterns in response to environmen generations.'2 Methyl groups from 5-adenosyl-methionine tal exposures. To our knowledge this is the first study to report (SAM) are needed for DNA methylation. Diets low in sources global D N A hypomethylation in umbilical cord serum signifi o f methyl groups can lead to global DNA hypomethylation by cantly associated, in a dose-response fashion, to in ucero expo impairing synthesis of SAM .11 Folic acid depleted dietary intake sure to tobacco smoke. These resales are consistent with Brecon has been shown to lead to global DNA hypomethylation in ani et al." who reported association between tobacco smoke and mal studies. The loss o f global DNA methylarion can be reversed DNA global hypomethylation, albeit in a different life stage and if folate is added to che diec on or before che ninth week. If folate 4 Eplgenetics Volume 5 Issue 6 p. 20 | Figure 2. (A) Linear relationship between the natural log o f cotinine in cord blood serum and th e global DNA m thylation index in 30 newborns w ith ; high (11), low (11) and very low (8) cotinine serum levels. (3) Linear relationship between the natural log ofPFOA In cord blood serum and the global | DNA m thylation index in 30 newborns. (C) Linear relationship between the natural log o f PFOS in cord blood serum and th e global DNA mthylation | Index in 30 newborns. www.landesbioscience.com Epigenetics 5 Table 3. Quantification o f serum Y chromosome human DNA(ng/ ul) and serum total human DNA (ng/ul) obtained from umbilical cord blood o f seven male infants, baltim ore three study Q uantifier Y hum an DNA Ing/pl) Q uantifier human DNA (ng/pl) Y human DNAThuman DNA Sample Levels Mean Levels Mean Ratio 1 4.38 4.32 2.75 2.71 4.26 2.66 1.6 2 0.18 0.16 0.08 0.09 0.15 0.10 1.8 3 0.20 0.21 0.02 0.02 0.22 0.02 8.7 4 3.93 4.08 2.94 2.80 4.23 2.66 1.5 5 0.08 0.08 0.01 0.01 0.08 0.01 6 0.46 0.44 0.05 0.03 15.5 0.42 0.01 14.3 7 0.11 0.12 0.04 0.04 0.14 0.04 3.4 is introduced afterwards, DNA mthylation loss and progres sion to cancer can be stopped, but not reversed.'"1 Interestingly, preconceptional exposure to famine during the Dutch Hunger Winter o f 1944-1945 was recently shown to be associated with hypomethvlation o f the insulin-like growth factor 2 {IGF2) and (INSIGF) gene six decades later.3' 16 Epigenomic alterations associated to in utero exposures may thus have life-course effects. The epigenome can be visualized as an interface between the dynamic environment and the inherited static genome, configured during development to shape the diver sity o fgene expression programs for different cell types by a highly organized process.'7 Cell differentiation during embryonic-fetal development involves different epigenetic processes which, if altered, may affect either somatic or germ cells.'* Epigenetic alter ations can occur in somatic cells at different stages o f life, from fecundation to adulthood, and when germ cells are affected, such changes can even be passed on to future generations.1' Physical, biological and chemical factors, as well as social factors, such as maternal care, modify the epigenome. Thus, exposures to envi ronmental agents such as tobacco smoke in utero may influence susceptibility to disease throughout the life-course, via epigenetic alterations, which can be used as biomarkers of exposure and risk. Epigenomic biomarkers may lead to advancements in the fields o f developmental diseases biology and molecular epidemiology, where the search for molecular biomarkers o f exposure-related effects that can be used for screening, regulatory and risk predic tion purposes has proven elusive. The main challenge has been to identify molecular biomarkers that are part o f the downstream effects triggered after the fetus is exposed to the toxic environ mental contaminant. Global D N A mthylation status is, probably, not a specific biomarker o f in utero exposure to cigarette smoke and PFCs only, but to many other environmental exposures, such as air and water pollution, and dietary intake as well. In addition global DNA hypomethylacion is known to be associated with a series of chronic diseases, including diabetes, bipolar disorders, obesity, schizophrenia and cancer.'11'1 Thus establishing a link between exposure and disease is not as important as detecting the loss of mechylation to ascertain risk and determine therapeutic and behavior modification alternatives. In many cases, epigenetic modifications are reversible, thus providing an opportunity to reverse the chronic disease process and understand the impact o f lifestyle choices on chronic disease susceptibility and risk.'12 In the case o f PFOS and PFOA, no studies oil DNA methylation have been reported; however, exposure tn peroxisome proliferators have been found to cause global hypomethylacion in che rat liver.'1 This study has several limitations. The sample size limited the possibility o f performing multivariable regression analyses with several predictors simultaneously, which may have eluci dated associations o f global DNA hypomethvlation levels in cord serum with maternal and paternal variables. The detection limit for the Imprint Methylated DNA Quantification Kir is 5 n g o f methylaced DNA and input DNA used may be as low as 10-200 ng. The sensitivity of the ELISA assay, limited by antibody affinity and substrate availability may have led to a slight under quantification of global DNA methylation levels. However this sensitivity issue should not affect the relative difference global DNA methylation across cotinine or perfluorinated alkyl levels in serum. Finally, lack o f information on other environmental factors known to affect D NA methylation status, such as folate, arsenic and cadmium levels may be confounding the associations reported in this study, Both environmental and dietary factors have been shown to modulate global DNA methylation levels, yet their combined effect has not been elucidated." These questions should be pursued in follow-up studies. Altogether the findings of this prool-of-principle study sug gest that differential global D NA methylation levels may be used as a bio-dosimeter o f fetal exposure and o f exposure associated biological effects on the fetus in cord serum. Furthermore, if the association between global DNA hypomethylation and cotinine levels in cord serum is validated in a larger cohort, smoking ces sation effoccs among pregnant women should be strengthened. Finally, global biomarkers o f epigenomic alterations may be use ful biomarkers for chronic diseases related to persistent environ mental exposures. The results o f this study suggest that a detailed examination o f these possibilities is warranted. Materials and Methods Subject selection. Thirty (30) subjects were sampled for this proof-of-principle study from 113 subjects who met the crite ria of having been born term (>37 weeks gestacion), of normal weight (>2,500 grams), and having cotinine levels and adequate amounts of serum. The sample stratified on cord serum coti nine levels: (a) High cotinine-Serum cotinine levels indicative o f recent maternal active smoking (>10 ng/ml) regardless o f smok ing history; (b) Low cotinine-Serum cotinine levels considered 6 Epigenetics Volume 5 Issue 6 to be clearly within a passive smoking range (1-5 ng/ml) among DNA Quantification Kit, Sigma Aldrich). The M D Q l kit is a mothers reporting no smoking during pregnancy; (c) Very tow high-throughput, molecular biology kit, which uses a 96-well cotinine-Serum cotinine levels consistent with nonsmokers (<1 plate formac to provide accurate differential global DNA mth ng/ml) among mothers reporting no smoking during pregnancy. ylation absorbance readings with as little as 50 ng of genomic O nly term and normal weighc newborns were selected to avoid DNA. Two microlicers (pi) of DNA at a concentration of 100 potential interactions between epigenomic levels and factors ng/p.1 were diluted with 28 Jil o f lysis and binding buffers and leading to pre-term birth and or low-birthweight, which may incubated at 60C. The samples were incubated with capture and confound the association between global DNA mthylation and detection antibodies and absorbance was read at 450 nanometers. cotinine or PFC levels in serum. Sampling was not done at ran Quantification of global DNA mthylation was obtained from dom but rather the nonsmokers were selected from among those calculating the amount o f methylated cytosines in the sample (5 that had both, no history o f smoking and the lowest cotinine val mC) relative to global cytidine (5 mC + dC) in a positive control ues to increase statistical power. Sampling was blinded to levels that had been previously methylated. All samples were analyzed o f polyfluorinated chemicals PFOS and PFOA. in duplicate. Subjects were selected from the Baltimore THREE (Tracking Y chromosome quantification. The Quanti filer YHuman Male Health Related to Environmental Exposures) Study, a cross-sec D NA Quantification Kic (Applied Biosystems) and Quantifier tional study o f newborn deliveries at the Johns Hopkins Hospital Human DNA Quantification Kit (Applied Biosystems) were used in Baltimore, M D, designed to determine cord blood levels of a to quantify the amount of total amplifable human DNA and Y number o f environmental chemicals, along with their relation Human Male that was present in 8 cord blood DNA samples ship to thyroid hormone status and birth outcomes. Data from obtained from male infants. The quantification assay combines this study have been reported in several peer-reviewed publica three 5' nuclease assays, namely; targec-specific human DNA tions.'-'1The study received approval from the Johns Hopkins assay and target specific Y human male DNA assay, plus an inter Medicine Institutional Review Board and was determined to be nal PCR control (IPC) for both chemistries. The total human HIPAA exempt. target in this assay is a 62-base Human Telomerase reverse tran Umbilical cord blood samples were collected immediately after scriptase gene (hTERT) located at 5 p l5-33, which is detected by birth using the Witter cord cradle (Witter et al. 2001), and stored a TaqMan MGB probe labeled with FAM dye. The male target, at 4C for <3 hr before further processing. O f these, 41 samples the 64-base sex-determining region Y (SRY) located at Ypll.3, had insufficient cord blood volume for laboratory analyses, leav is detected by a TaqMan8 MGB probe labeled with FAM dye. ing a total o f 300 samples available forchemical laboratory analy The IPC, 130-base PCR control, a synthetic sequence not found ses. Once collected, umbilical cord and cord blood samples were in nature, is detected by a TaqMan* MGB probe labeled with placed in the Labor and Delivery Suite refrigeracors. Within a VIC dye. The targets are measure by Forster-rype energy transfer few hours o f birth, samples were collected and transported by (FRET) technique. The ratio of human male DNA to human study personnel to a laboratory at the Johns Hopkins Bloomberg DNA in each sample is calculated by dividing rhe amount o f Y School of Public Health for processing. Umbilical cord samples human male D N A by the amount of human DNA present in were flash frozen. Blood specimens were centrifuged at 1,000 g each sample. This test is enriched to show a high male signal for 15 minutes, and serum was aliquoted into 2m L polypropylene among a large amount of female DNA. Therefore, the ratio of cryovials. Tissue and blood samples were then stored at -80C. human male D N A to human DNA that rules out the presence of Frozen samples were transferred on dry ice to laboratories at female DNA can be equal to one or higher. the Centers for Disease Control and Prevention in Atlanta for Statistical analysis. Correlation analyses, Welch t-tests and cotinine analyses while other aliquots were archived. A total of Wilcoxon rank rests were used to evaluate the differences in the 286 samples had sufficient volume for measurement o f cotinine global DNA mthylation index across levels o f exposure to ciga was conducted using positive-ion atmospheric pressure chemical rette smoke and PFCs in cord serum DNA; and to evaluate the ionization tandem mass spectrometry as previously described.1'1 differences in the global DNA mthylation index across levels of Likewise the C D C laboratory analyzed serum samples for PFOS exposure to cigarette smoke in cord tissue DNA. Bivariate (unad and PFOA, using negative-ion TurboIonSpray ionization, a vari justed) linear regression models on DNA mthylation index ant of electrospray ionization, tandem mass spectrometry.20 were carried out to evaluate relationships to cotinine, PFOA and D N A isolation from cord blood serum. O f 286 samples PFOS and the natural logs o f cotinine, PFOA and PFOS as well analyzed by the C D C , 113 met the inclusion criteria (above), of as smoking status. Other potential Independent variables were which 30 one ml cord serum samples were digested with 50 ug/ explored including maternal age (linear and categorical), mater mL proteinase K (Boehringer Mannheim, Germany) in the pres nal prepregnancy body mass index (BMI) (linear), sex, race and ence of 1% sodium dodecyl sulfate (SDS) at 48C for 3 days, fol gestational age. On the basis of this exploration, separate multi lowed by phenol/chloroform extraction and ethanol precipitation variate models were constructed. Models were constructed using and finally dissolved in 30 (iL o f LoTE (2.5 mmoi/L EDTA and Stata version 10. 10 mmol/L Tris-HCL) as previously described.21 Global D N A mthylation assays. The global DNA mthyla A c k now led gem ents tion levels in umbilical cord serum samples were obtained with This research was supported in part by the NCI 5U01CA13-8, an ELISA based commercial kit (M D Q l, Imprint Methylated It also received support from the .Maryland Cigarette Restitution www.landesbiosdence.com Epigenetics 7 Program Research Grant given ro rhe Johns Hopkins Medical Institutions and by funding from the N IO SH Education and Research Center for Occupational Safety and Health at the Johns Hopkins Bloomberg School o f Public Health (#T42CCT310419). R.U.H. was supported in part by grant 1ROIES015445 o f the National Institute o f Environmental Health Sciences (N1EHS). 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Hcrranz M, Esceller M. DNA mcthylation and histone modifications in patients with cancer; potential prog nostic and therapeutic rarcers. Methods Mol Biol 2007; 361:25-62. 43- Pogribny JP, Tryndyak VP, Boureiko A, Melnyk 5i. Bagnyukova TV, Montgomery B, ei al. Mechanisms o f peroxisome proliferator-induced DNA hyponv echylaiion in rat liver. Mucar Res 2008: 644:1723. 44. Lee OH, Jacobs DR Jr. Porta M. Hypothesis: a unify ing mechanism for nutrition and chemicals as lifelong modulators of DNA hypomethylation. Environ Health Perspeet 2O09; 117: 1799-802. 8 Epigenetics Volume S Issue 6 ISOON SNIV1NOO