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NEWS FOCUS I)ioxin 'I'oxicity: New Studies Prompt Debate, Regulatory Action New data on dioxin's effect on humans, a clearer picture of the cellular events it precipitates, and new animal toxicity studies may provide EPA with a firmer basis for regulation Davld J. Mes*oN C&EN Washington Of all the chemicals that have been tossed into the cal- drort of public anxiety, 2,3,7,8-tetrachlorodibenzo-p-di- oxin, TCDD, has achieved the most notoriety and evoked the greatest fears for the longest period of time. Beginning with herbicide use during the Vietnam War, propelled by Love Canal and Times Beach, and sus- tained by reports of contamination from city incinera- tors and paper mills, TCDD essentially defines chemi- cal contamination for most people. 7f:DD was first implicated as the culprit responsible for ilInesses among chemical industry workers who produced 2,4,5-trichlorophenol. However, the chemical was thought to be only of industrial concern until the mid-1970s. In 1976, an explosion at a chemical plant spread from I to 4 lb of TCDD over the residents of the town of Seveso, Italy. High TCDD levels were recorded in people from the area. Shortly afterward, many Viet- nam veterans began wondering if their range of illness- es co dd have been caused by exposure to the herbicide agent orange which ~ as known to be contaminated with TCDD. When TCDD was found to be part of the highly re- ported and emotionally charged hazardous-waste leak- age that resulted in evacuation of the community of Love Canal, Niagara Falls, N.Y., in 1980, and evacuation and purchase by the federal government of the entire town of Times Beach, Mo., in 1983, it became firmly im- pre:sed in people's minds that here was a chemical prob- lem of elephantine proportions. But just as the four blind men each described an elephant differently because they werrn each touching different parts of the animal, so dif- ferent scientists, government officials, corporations, and environmental activist groups describe the_ hazards of TCDtr) differently. It is only recently~that a clearer pic- ture of this creature has emerged, and veile it may not be the mammoth once believed, it is no Inouse either. Several events have come together over past months that have helped focus the picture. One is a symposium Receptor-mediated TCDD action requires several steps 1. TCDD attachn to 2. 3evere7 activated coralezes attach Ah rec®ptor and to spedfie sicA on DNA before translocaErg ptntoin mesaerx,pr RNA q transcnbed TCDD \ Nucleus 3. tibae®agAr RNA carries code for cytochronw P4501 AS or other enzymes In tlv cell, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binds strongly to a soluble intracellular protein designated the Ah (for aryl hydrocarbon) receptor, which then binds to a trans- locating protein that carries the "activated complex" into the cell nucleus. Several activated complexes bind to spe- cific DNA sequences, distorting the DNA chain. Ensuing events lead to transcription of messenger RNA that codes for cytodxome P4501A1 or other enzymes held at the Banbury Center at Cold Spring Harbor Labo- ratory, New York, in October that took a critical look at the state of the molecular science of TCDD. The conclu- sions reached there are extending far beyond research laboratories. Another is the release of significant epide- miological research data involving people exposed to TCDD aimed at determining whether or not they are suffering illnesses as a result. The EnvironmentaI Protec- tion Agency, some say in response to these events, has Augtrst 12. 1991 C&EN 7 ®

News Focus und:ei°taken an important review of TCDD toxicity that may lead it to change its policies on carcinogenicity. Ivluch of TCDD's dreadful image stems from its label as "the most toxic synthetic chemical known to man." It is widelv conceded that this label is attributable to ear- ly research that found that as little as 0.6 µg per kg body weight could kill 50% of guinea pigs exposed to TCDD. Research by Dow Chemical scientists in 1978 found that the chemical was also a potent carcinogen in rats. These facts, repeated in virtually all newspaper and television reports about TCDD, made the chemical the most iieared of all contaminants. Subsequent decisions to lower TCDD concentration in the environment led to the banning of products such as 2,4,5-trichlorophenoxyacetic acid, a herbicide con- taminated with TCDD. Production of other products, especially those that used 2,4,5-trichlorophenol as a precursor, was stopped. When it was discovered that municipal and hazardous-waste incinerators were large sources of TCDD in the environment, new controls were implemented to limit TCDD emissions. Use of leaded gasolines was also found to be a prime source of environmental TCDD, and EPA's phaseout of leaded gasoline has also resulted in reduced emissions. Based on its current risk assessment model, EPA has calculated that the tolerable daily intake of TCDD for humans is 0.006 picograms per kg body weight per day. This level would supposedly result in a one in 1 mil- lion chance of excess cancer from TCDD. But this level is far, far below the 1 to 3 picograms per kg body weig;hC per day that people are estimated to be actually ingestiing. Other nations, using different risk assess- ment models, have calculated the tolerable intake level much higher.. Germany uses 1 picogram, the Nether- land:s ~.1, and Canada and the World Health Organiza- tion use 10 picograms per kg body weight per day. Because health problems associated with TCDD expo- sure fitst came to light as the result of chemical indus- try accidents in the 1950s, the industry has always been a target of blame for environmental contamination. Dow Chemical, Monsanto, ::nd six other chemical com- panies that made agent orange for the federal govern- ment during the Vietnam War were the defendants in a major lawsuit that was settled when the companies agreed to pay S180 million to vet• erans claiming illnesses resulting from exposure to the herbicide. TCDD has subsequently been at the root of thousands of injury lawsuiis, including cases filed by people who lived at Times Beach and I ove Canal A significant decision was handed down this summer when the Illinois Appellate Court of the 5th District overturned a 3r16.25 million punitive damage verdict against Monsanto (C&EN, June 24, page 6). The jurors had found no injuriesr among people claiming to have been injured by dioxin expo- sure following a train accident, 8 Augus 12. 1991 CdEN but decided to punish Monsanto anyway. The avoe:,.is court judges ruled that if there are no injuries, F ~ve damages cannot be exacted. A request to have , ap- peal reheard was also rejected. Some attorneys specializing in personal injury cases believe this decision may be a turning point for TCDD and other chemical injury cases. David G. Owen, law professor at the University of South Carolina, for in- stance, says: "The decision may stand as a declaration ot judicial intolerance to the use of courtrooms as arenas for subjecting U.S. industry to political and ideological persecution." David Snively, litigation counsel for Monsanto, has already seen an actual drop-off in dioxin litigation. The company ceased making chlorinated phenols a decade ago, and Snively says about the only relationship Mon- santo now has with TCDD is some research. "There has been a fall-off in litigation involving Monsanto, and we have no active lawsuits," he says. He has noticed that new lawsuits seem to be cropping up in the paper and pulp industry. The paper industry was surprised in 1985, when TCDD was discovered in the effluent and sludge from paper mills. It was found that chlorine bleaching of the pulp caused TCDD formation. Claims soon arose that TCDD was leaching into children's milk cartons, and EPA found itself faced with a lawsuit brought by the National Wildlife Federation and the Environmental De- fense Fund demanding regulation of TCDD in the mill effluent. Although concerns about human health h. .:rds from paper products have subsided, the environmental concerns have spurred paper manufacturers to treat ef- fluents to reduce TCDD and to look into process chang- es to decrease TCDD production. The industry uses about 14% of all chlorine produced in the U.S. and con- centrations of TCDD in the effluents are in the parts- per-quadrillion range. The Chlorine Institute, whic'r represents the chlor-alkali industry, is very worrie( about this problem, but maintains that the parts pe trillion and lower concentrations of dioxins and furan generated by papermaking do not pose a health or en vironmental threat. However, the Chlorine Institute was one of the spon TCOD tox3cfty varies great#y among species i Speck$ LOe. (u6 8- k0) ! i Guineapi® IkMnbc 0.6-2.5 I b ( I Roo 22-320 ~ I M, k.y <70 I Rabb@t 115-275 ~ Mau" 114-2ao Dog > 104'<3000 Macrster 1150-5000 I Saaw EmrornmmW Fraecian Agiw..y sors of the Banbury Center meet- ing. The conference, which brought together 38 invited scien- tists from the U.S. and Europe, was actually the second meeting held to discuss TCDD; the first was in 1985. In addition to the Chlorine Institute, the meeting was cospon- sored by EPA and FDA. All scien- tific aspects of the issue were laid out at the conference, and by the end, there was some general agree- ment on several issues. Michael A. Gallo, associa an of research at the Robert _od h M 1 S h 1 'v Jo nson c c oo in . ew di e a Jersey, was one of the conference organizers. "We met for four days t•\ \ Vm1polmw

and we reviewed all the science, and we reviewed a lot of the math- ematical and biological models that went into it. We came away with th.e idea that the primary events in arid around dioxin were related to this thing we call the Ah receptor." Gallo says the meeting did clarify i:he scientific issues in that "it helped focus the thought patterns of where a lot of people want to go over the next year, and I think it foi:used where [EPA Administra- tor] Bill Reilly and EPA have to put their energies." Overshadowing everybody's concern over TCDD is that, despite the compound's obvious toxicity to animals, no clear-cut human health problems were associated with TCDD exposure, even though a number of, human exposure inci- dences were known. Epidemiolog- ic >tudies undertaken to identify any major health problems always h,ad so manv shortcomings that researchers could not say whether TCDD had an effect or not. This year two stud- ies have brought the answer closer. The Air Force Ranch Hand study, begun in 1978, is the oldest study of people heavily exposed to TCDD arid the most controversial. An ongoing study, it con- tinLies to draw severe criticism. Study participants are members of Operation Ranch Hand, the group that ac- tually handled and sprayed agent orange over the fields and forests in Vietnam from 1962 to 1971. Agent orange was used to defoliate forests and kill some crops in Vietnam for the protection of U.S. forces. It was a 50- 50 cc)mbination of the herbicides 2,4,5-T and 2,4-D and was contaminated with about 2 ppm or less of TCDD. The original study involved 1242 Rand Hand person- nel, although the actual number of participants varies somewhat from examination to examination. The strongest critic of the Ranch Hand studies has been, the American Legion, which represents U.S. vet- erans who served in Vietnam. The American Legion says the Ranch Hand studies are not designed well enough to see excess diseases because of too few pa.rtic- ipanl:s and that the classification for exposure was im- properly done. It further charges that the Air Force has actually suppressed adverse health effects findings. De- spite the Air!Force's claims of no significant illness such as caricer or reproductive effects, the legion, with back- ing from some researchers it has supported to do its own reviews, believes the Ranch Hand personnel have suffered from TCDD exposure. No one has doubted that those in the Ranch Hand study were exposed to TCDD. In the Air Force's latest report, issued in March, the blood levels of TCDD were measured in 866 Ranch Hand veterans and 804 compar- ison veterans for the first time, and those levels were correlated to any health effect they could find. Values for TCDD in'blood serum ranged from 0 to 618 ppt in Houk: improve risk assessments Ranch Hand personnel, but the median value was 12.8. Median value of the comparison group was 4.2. Aside from some other, very minor correlations, the report found a significant increase in body fat and diabetes that correlat- ed with TCDD concentration. This was a surprise, according to William H. Wolfe, chief of the ep- idemiology research division of Armstrong Laboratory at Brooks Air Force Base in Texas, which is the lab responsible for Operation Ranch Hand. "This obviouslv needs a closer look," Wolfe savs. "We are doing another examina- tion of the same group starting early next year. We are changing and extending our physical exam- ination tests to look criticallv at the diabetes and to get more an- swers." Wolfe adds that this find- ing, while serious, did not get much attention perhaps because "it is not one of the diseases that people had a vested interest in." The American Legion criticisms of the March Ranch Hand report did not even mention the diabetes finding. Wolfe admits that some of the criticisms of the Ranch Hand study are valid. The small sample size is a problem for identifying increases in diseases like rare cancers. "These limitations were all laid out at the beginning," Wolfe says; "we just got all the Ranch Handers there were." Statistically, he says, "the study has only a S0°c ability to detect a doubling of rare cancers like soft tissue sarcoma. And that's too low." For detecting a rise in all cancers, the odds are better. "If we take all cancers to- gether, we have a 90% chance of detecting a 501'c in- crease," he says. The complaints that data have been deliberately withheld are groundless, Wolf says. He points out that all the data go through a review committee that makes recommendations for additions or changes, which are taken care of before the report goes to the Surgeon General. Given the advisory committee oversight, Wolfe does not see how data could be left out, especial- ly since Sen. Tom Daschle (D.-S.D.), concerned that the Ranch Hand study might be getting inappropriate ad- vice, changed_the board composition so that 30% of its members were nominated by veterans groups. One of the most emotional issues of TCDD has fol- lowed in the wake of the Ranch Hand study. That is, whether other soldiers who served in Vietnam suffered from exposure to agent orange and the dioxin that con- taminated it. Study after study by the government have shown that men and women serving in Vietnam were not exposed to high levels of TCDD. The Centers for Disease Control tried more than once to find a correla- tion between Vietnam service and health problems or blood serum dioxin concentrations and could not iind one. The Department of Veterans Affairs also has stud- August 12. 1991 C&EN 9

News Focus ied veterans and concludes that agent orange caused no health probleats. The most recent report came from Han K. Kang, head of the Office of Environmental Epide- miology at DVA. Published in March, Kang's study measured blood serum TCDD levels in 36 veteran5 who,- from as best as could be determined from records of agent orange spraying and troop positions, might have been exposed to the herbicide. "We tried to come up with an ex- posure assessment based on this," Kang says. "We couldn't find any increased exposure. The difference between their body burdens of TCD,D was not signif- icant" compared with the control group. ]Kang says DVA is fin- ished 'with efforts to determine TCDD levels in Vietnam veter- ans. "We have done all we can in dealing with the veterans," he says. "'We are satisfied with the conclusion that there is no significant elevation in TCDD levels." One striking finding by Kang is that the body bur- den of TCDD in the population appears to be falling. The samples he measured were collected in the early 1970s by the National Human Adipose Tissue Monitor- ing Program. Kang reported mean background levels of about 12 ppt for these samples. More recent work, such as that done by CDC, found background levels of TCDD al: 5 to 7 ppt. "We are speculating that the envi- ronmental TCDD levels are going down, for whatever reason. Other countries, like Sweden, have also report- ed that: organic compounds, including TCDD, are de- clining," Kang says. Anot:her reason DVA does not feel it needs to do more srtudies is a change in administrative policy made in 19(Xby Secretary Edward J. Derwinski. In response to a law;;uit filed in California, the department has be- gun offering compensation to Vietnam veterans who suffer from diseases thought to be caused by agent or- ange exposure, even though no such link has ever been proven. The diseases currently accepted are soft tissue sarcoma, non-Hodgkins lymphoma, and a nerve prob- lem called peripheral neuropathy. The e;:)idemiology study likely to carry the most weight in the TCDD discussion was published in Janu- ary by Marilyn Fingerhut and her colleagues at the Na- tional Institute for Occupational Safety & Health. Her retrospective cohort study of cancer mortality included 5172 chemical plant workers from 12 companies who had worked in areas making products that were contam- inated wi.th TCDD. Generally they worked at piants that produced'. or used 2,4,5-trichlorophenol. Of these work- ers, 1052 had died, and the study is based on data on their cause of death. Fingerhut also had blood samples taken firom 253 workers from two plants for measuring TCD® has several toxic effects D"th wss&V syrwmr" Ttryrt* a"+y ; 8piefdc atOpRy Te~SaAw strophy tJma .rMarpsr"art, tstQy d.poMta, n.ar,air Hy{erplwa: gso4ria maroo.a, urtrs.rp traet, bN da,c! squ.',mw» n*eapiaod.= m.bortdan 9iar48, oerurrwwFa Olando CNaraat't: Mypaplaoia, htrperlccrsfo.k, alt.nd p4mor"Man Terato"nolk carcho"Ouft kroru P*Vaw~w Ettzyrrss tro*=Wr+ Eiod+sn4aal .tf.eb seuM EmTOrvn.4i Prot.aSm, AvKtr serum TCDD levels. The part~ pants were divided into grc that had less than or more th.~, one year of exposure to TCDD- contaminated materials. A sepa- rate analysis was done of indi- viduals who were exposed more than 20 years ago. John C. Bailar III of McGill University said in an editorial appearing in the same issue of the New England /ournal of .1,1edi- cine as Fingerhut's study was published, "Parties on both sides of the continuing debate about the regulation of dioxin exposure will no doubt cite this work in support of their positions." And he was right. Among the significant conclu- sions reached by Fingerhut's group is that, for the group that was exposed to TCDD for longer than a year and with more than 20 years' latency, deaths from cancers of all kinds combined were 46% higher than for the general population. "This is an unusual finding for chemical workers," Fingerhut explains. "Chemical worker studies to my knowled have not shown that. In the few I'm aware of that sec excess in all cancers, the excess has been accounted tvr by a single-site cancer, say lung cancer." Because of the problem of multiple exposures to chemicals among plant workers, Fingerhut explains, TCDD cannot unequivocal- ly be said to be the cause for this observation, but "it ap- pears to us that the most likely explanation is the expo- sure to chemicals contaminated with TCDD: " "We showed in this paper a striking correlation be- tween serum levels of TCDD and duration of expo- sure," Fingerhut says. Consequently, it will allow fol- lowup studies to assume that workers of longer dura- tion will have higher levels of TCDD in their blood. It is also of note that, with one exception, the Fingerhut study did not find excess cancer deaths from any one type of cancer, including non-Hodgkins lymphoma, which in the past has been associated with TCDD expo- sure. The Fingerhut report is inconclusive on the issue of TCDD's causing soft tissue sarcoma. Some say her finding of three deaths when less than one was predict- ed proves the connection. Others say the numbers are just too small to be meaningful. "As time goes on, re- analysis of this study group will be more powerful and it may be that the interpretation will become more clear," Fingerhut says. To Vernon N. Houk, director of the Center for Envi- ronmental Health & Injury Control at the Centers for Disease Control, the issue is mostly decided. "I beli, that a conservative interpretation of the Fingerhut st is that if dioxin is a human carcinogen, which I am ~- suming it is, it is a relatively weak one and is a carcino- gen only at extraordinary doses." Houk notes that Fin- gerhut's study agrees with results from the Ranch Hand 10 Augatt t2, 1991 C&EN

'i°CDD antiestrogen effect has tumor-fighting potential jkfter more than 30 years of experi- tsnce, there is little doubt that 2,3,7,8- totrachlorodibenzo-p-dioxin (TCDD) is a clangerous chemical to have in the en- vironment. Still, approximately 10 years ago, a few scientists took note of one effect of TCDD in rats and have since been pursuing an interesting and poten- tially valuable line of research. As Michael A. Gallo, associate dean for research at the Robert Wood Johnson Medical School in New Jersey, tells it, it all started with a Dow Chemi- cai study , by Richard J. Kociba, pub- li;ahed ab®ut 13 years ago. The study st9owed an increase in liver cancer in r2ts exposed to TCDD, "but at the ur,me time there was a remarkable de- crease in spontaneous breast tumors ar4 sp~srteous uterine tumors," Ca!- lo says. He and others began looking inlo this effect, wondering if TCDD, which seemed to be reacting with a harmoneiike receptor, might be an arr tiesfrogen. If it was, and could sorne- hcw be controlled, there was a poten- tial for reducing women's risk of estro- 13en-depend®nt breast cancer. Galio says early tests in mice cor} fimned ttiat:TCDD did somehow prevent estrogen from doing its job. "One ex- periment to measure estrogen activity is to inject estrogen into an immature mouse. The uterus will then mature very rapidly. So you end up with an im- mature animal with a mature uterus. We did it the other way. We injected immature animals with TCDD (an anti- estrogen). The untreated mice went on to develop normal uteruses and the an- imals given TCDO had immature or- gans." It was clear TCDD was prevent- ing estrogen from acting. There are two ways this might occur, The first is that the TCDD-receptor complex is actually inducing production of an enzyme that metaboiizes estradf- ol. Gallo says twe is some evidence of this, but that it alone is probably not enough to account for TCDD's ob- serve+d antiestrogenic effects. Gallo and collaborator Steven H. Safe, of the departrnent of veterinary medicine at Texas A&M UnNerstty, are working on the assumption that the TCDO receptor Is somehow Interfering with production of the normal estrogen receptor in cells. "We see changes in the messenger RNA for the estrogen receptors," he says. "It's decreased. study Cn that persons exposed at moderate levels do not seem to be having excess cancer mortality. He hopes that this, as well as additional research will be used in the fu- ture tc make better risk assessments. "The federal gov- ernm.ent has spent more than $400 million to research this compound, in addition to what the companies have spent. If we don't use that new information to modify or supp:)rt our views on dioxin, then why did we do it?" The next body of knowledge coming to bear on TCDD's toxicity will be the result of research on ani- mals and at the molecular level. One of the most signif- icant realizations of the past few years is that TCDD cannot be considered by itself. Molecular biology has shown that the action of TCDD in a cell is receptor me- diated and that there are a number of dioxinlike com- pound: that can all have toxic effects. This receptor-mediated action for TCDD was first dis- covered in 1976 in Alan Poland's lab at the University of Wisconsin, Madison. Poland and others injected low lev- els of TCDD into animals and found that one of the first things that happened was the induction of a particular cvtochrome P450 microsomal enzyme that oxygenates substrates, such as the carcinogen benzo[a]pyrene, that accumulate in fatty tissues. The response depended on the dose and had a great deal of potency. Researchers also noted that other planar chlorinated dioxins, dibenzo- And ttv actual estrogen receptor pro- tein is decreased after TCDD treatment at very low doses." Safe adds that "TCDO also seertu to block other stim- ulatory agents that make breast cancer cells grow." A common drug used currently as a palliative breast cancer therapy, tamox- ifen, has some side effects and, Gallo says, about 15 % of the patients put on the drug becorrte susceptible to tumors again after f'rve or six years. "if we had a drug that didn't allow the estrogen re- ceptors to be syMhesized in these can- cer victims, we wouldn't have to worry about blocking their action," he ex- plains. Thus, if TCDO does functionally block formation of estrogen receptors, there is ciinical potential, Of course, the toxicity of TCaD is such tltisi it would not be used as a drug. However, Safe says they have ab ready synthesized relatively nontoxic TCDD analogs to see if those com- pounds produce the same responses. "Other dioxins and dibenzofurans that we've made have less toxicity but they still seem to have these potent anties- trogenic effects. Not as potent as TCDO, but still pretty good," Safe says. furans, and polychlorinated biphen,vls (PCBs) could elic- it a similar response, although not so strong as TCDD. The evidence, found first in chicken embryos and tis- sue culture, then in rats and other animals, looked like classic receptor-mediated transduction, very similar to the way steroid hormones are made in cells. The recep- tor was recognized as a soluble intracellular protein and was designated the Ah-for aryl hydrocarbon-recep- tor. One experiment that clinched the presence of this receptor was that mice, inbred to be genetically without the receptor, do not respond with enzyme induction when treated with TCDD. Although it is impossible to say for sure, all evidence points to only one receptor for TCDD, one that begins all TCDD cellular activity. So TCDD gets into the cell, where it binds strongly to the Ah receptor. It was discovered, however, that this wasn't enough to generate induction of P450. Subse- quent research identified another protein, a translocat- ing factor, necessary for the complex to pass from the cytoplasm into the nucleus. This protein has just recent- ly been cloned by Oliver Hankinson and coworkers at the University of California, Los Angeles, department of pathology and the Laboratory of Biomedical & Envi- ronmental Sciences at UCLA. Once inside the nucleus, it seems that several-pos- sibly as many as four or five-of these complexes attach August 12. 1991 C&EN 11 EFEMMMMORM .. _ .._ - ._~ ~.::. .--- ~ ZOS#+!3547:RZ32

Alews F6Cus to a specific sequence on the cell's DNA. This, accord- ing to James P. Whitlock Jr. at Stanford University School of Medicine's department of pharmacology, dis- torts the DNA chain, allowing attachment of other binding proteins. These events lead to the transcription of messenger RNA that forms the cytochrome P450 or other enzymes that might be induced by TCDD. These steps, too, are typical of a steroid induction in cells. TCDD is now known to affect several genes. The gene for the original cvtochrome, which is now called P4501A1, and a second cvtochrome, P4501A2, are just two examples. Others include a gene for glucuronvl trinsferase,' glutathion-S-transferase, and aldehyde de- hydrogenase. Researchers have identified some other genes activated by the TCDD complex but have not fully characterized the mechanisms yet. Although the enzymes these genes produce are not believed to have anything to do with TCDD toxicity, it is clear that the TCDD complex is the mechanism through which TCDD exhibits a toxic effect. Studies done with the Ah-receptor-defective mice showed that all 1.he different toxicities of TCDD measurable in mice-chloracne (in hairless mice), porphyria, cleft pal- ate formation, teratogenesis, even death-were limited to the mice that had the high-affinity receptor. Struc- ture activity relationship studies also confirm this, ac- cording to Whitlock. The more potent the ligand for the receptor, the more potent the biological response. TCDD is the strongest binding of all the dioxins and dioxinlike compounds. The conclusion is that all the biological and toxic effects of TCDD and dioxin-related compounds are mediated through this one receptor. Exactly what the receptor is, is still unknown. So far, its structure has remained elusive, although some re- searchers are getting close. Scientists have noted, too, that small differences in the receptor occur among spe- cies. They know these receptors have different molecu- lar weights, but they don't know what that might mean with respect`,to affinity for TCDD. The Ah receptor is similar to the receptor that induces steroid hormones, such as estrogen. Thus, some scientists have put the Ah receptor in the same family as steroid hormone recep- tors, a.lthough others are convinced there are differences. On e scientist working on the receptor model is Ellen K. Silbergeld, adjunct professor of toxicology at the Uni- versity of Maryland, College Park. "TCDD's interaction with this receptor is very powerful," Silbergeld says. "Frorl my perspective, it must be doing something very impo;rtant in normall cell physiology. I take it as an as- surnption that somewhere there is a natusal compound that responds to the Ah receptor and transduces a set of eve;nts very important in normal cells." Silbergeld and other researchers point out that toxicology research has often found the unnatural ligand for receptors before it finds the natural compound. Some scientists believe that finding this endogenous ligand, which TCDD apparent- ly aamics, for the Ah receptor will be a central issue for TCDD research. The receptor was found for opiates be- fore the body's own endorphins were discovered, for ex- ample. The very high specificity that TCDD and other dioxin- like compounds have for the Ah receptor is key to the development of what is called toxicity equivalents, E cause other dioxins, dibenzofurans, PCBs, even ~ logenated naphthalenes bind to the receptor ar, ,'du cvtochrome P4501A1, it is assumed that they can cau the same range of toxic effects as TCDD. Because TCC has the strongest affinity, it is given a toxicity equivale cy factor of 1, and all others are being compared to EPA has adopted a list of factors for calculating toxicit,. of these mixtures of compounds. For instance, a pe tachlorodibenzo-p-dioxin with four of its chlorines the 2,3,7, and 8 positions is given a value of 0.5. C tachlorodibenzo-p-dioxins have a toxic equivalenetizero. The implications for measuring health effects of i these chemicals in humans and animals is significant. means that no longer can TCDD really be considere alone, but only as part of a potential problem. Accor ing to Linda S. Birnbaum, director of the environme, tal toxicology division of the EPA Health Effects R search Laboratory in Research Triangle Park, ti.C "There are suggestions that coplanar PCBs may be, fact, responsible for much of the toxicity equivalency human serum. In other words, in industrialized cou: tries, the background level of TCDD in people may ; 7 ppt, and if you add the toxic equivalencies of all tl other dioxins and furans, it gets up to about 30 ppt, you now add the toxicity equivalents of all the PCE you might say that people are actually walking arour with 100 ppt of dioxin equivalents in their body." While arguments will persist on what toxi °'ec these levels of contaminants have on people, t. .'.aj already significant data an their effects on fish an wildlife. Philip M. Cook of the EPA Environmental R, search Laboratory in Duluth, Minn., reports that trot in Lake Ontario have an average of 35 ppt TCDD. Ricl ard E. Peterson at the Environmental Toxicology Centt and School of Pharmacy at the University of W isconsi: Madison, reports that concentration of 65 ppt TCDD eggs of lake trout can cause 50% mortality. Peters, says newly hatched lake trout exposed to TCDD in t egg stage are the most sensitive to the lethal effects TCDD compared to any mammal, bird, or fish spec: ever investigated. Cook says, though, that TCDD co centrations were higher in the past. "The tendency h been for the concentration [in fish] to come down," i says. There are no archived fish samples for compar son, but Cook guesses peak concentrations occurre sometime in the 1960s. This observation agrees wit the finding by Kang that TCDD levels in humans ma be dropping too. One of the problems facing EPA is how to measur the buildup of TCDD in organisms as the contaminar moves up the food chain. The agency presently a: sumes a bioaccumulation factor of 5000 for TCDD leN els in water. This may be too low, argues Peter deFur, scientist working for the activist organization Enviror mental Defense Fund, who says EPA is using old dat. EDF recommends that the factor be raised to >a~ 50,000. On the other hand, Cook says their b. ,:st mate for bioaccumulation of TCDD in Lake Ontar: trout is on the order of 160,000. Though death is obviously the worst biological effe, 12 IApat 12. 1991 C&EN ® ~

of TCDI) and dioxinlike com- pounds, other problems are seri- ous as well. Cook says the levels of TCDD in Lake Ontario are probably leading to a lack of nat- ural reproduction in fish. At the U.S. Fish & Wildlife Service in East Lansing, Mich., Timothy Kubiak is investigating the re- productive effects of TCDD on mink, the species most sensitive to the compound after guinea pigs. He says by examining the toxicity of each of the individual congeners in mink, and, depend- ing on several'factors, TCDD may contribute to only about 5% of the total toxicitv, most of which comes from PCBs. Mink are a gooci species to study, Kubi- ak adds, because their sensitivity makes them a good sentinel for changing concentrations of pol- lutants. One toxic effect may be sexual Silbergeld: physiology of Ah receptor to a specific receptor in cells. Sec- ond is an evaluation of the sci- ence in various aspects of health effects, including carcinogenici- ty, reproductive effects, immu- notoxicologv, acute and chronic effects, and human epidemiolog- ical data. The final three items are a health research component, an ecological research compo- nent, and an exposure reassess- ment component." The entire project is supposed to be com- pleted by May 1992. Reconsideration of its cancer model represents a major break with tradition for EPA. The present linearized multistage model for carcinogenicitv does not work for receptor-mediated molecules because it does not al- low for a threshold below which cancer would not occur, Accord- ing to Farland, the EPA guide- lines, however, do permit the aberration.s in birds and animals from exposure to diox- inlike compounds. Theo Colborn of the World Wildlife Fund in Washington, D.C., is monitoring research on the effects of organochlorine compounds on wildlife, and she says t;hat researchers are reporting instances of her- maphrodi:ic offspring, such as male birds with oviducts, and abnoratal female-female pairings of birds. Colborn contend> that this is happening because TCDD and the dioxinlike compounds are messing up the estrogen re- ceptors on developing embryos at critical times. "One dose of TCDD to rats on day 15 of pregnancy, that's about when sexua! differentiation occurs, found a dose response in demasculinization and feminization of male offspring," she savs. All the information on animal toxicity and the data on molecu- lar chemistry are going to be taken into account during a ma- jor effort, now under way, that EPA is making to revise its pro- grams for regulating TCDD. Af- ter publication of'the Fingerhut study, artd, the scientific agree- ments that came out of the Ban- bury conference, EPA's Reilly told the agency it was time for a reassessment. William Farland, head of' EPA's Office of Health & Envi- ronmental Assessment for the Office of Research & Develop- ment, says, "The review includes five major activities. First is the evaluation of the biologically based dose response model for TCDD, taking into account the idea that TCDD is known to bind agency to change its model. "The 1986 cancer guidelines suggest that one ought to choose a dose response model with a biological rationale. TCDD provides a good exam- ple of a chemical [for which] a lot of good studies have been done, and we ought to be able to bring this infor- mation into our dose response analysis. So we think this is consistent with the agency's advice all along and is one of the best opportunities we have to put that advice into practice." EPA's Birnbaum is heading the agency's research on health effects. "Basicallv there are three areas we are trying to address here. First, we are trying to define the dose response curves, focusing on the most sensitive Birnbaum: sensitive indicators needed toxicological endpoints. Next, we are looking at enzyme induc- tion, specifically cytochromes P4501A1 and P4501A2. Finally, we are trying to find out where people are with respect to these responses." TCDD has many molecular ef- fects that can now be measured. Ligand binding to the Ah recep- tor, nuclear occupancy of the ac- tivated complex, cytochrome in- duction, and immunotoxicitv are examples. But each of these seems to happen at a different level of TCDD exposure. Birn- baum says EPA is focusing on the low-dose region of the re- sponse curves for these markers, looking for the most sensitive endpoints. When it comes to measuring effects of TCDD, can- cer may be a poor indicator. Birnbaum says it looks like im- August 12. 1991 C&EN 13

News Focus munotoxiciry may be the most sensitive indicator of TCDD effects, and this is clearly a problem. Cytochrome induction has been considered one of the easiest responses to detect. The P4501A1 enzyme can be induced in most cells and animals. Measured in terms of the action of aryl hydrocarbon hydroxylase, the cytochromes are important because they can actual- ly metabolize some environmental chemicals into sus- pected carcinogens, Birnbaum says. "These cy- tochromes are present in high concentration in the liv- er in response to TCDD and related chemicals," she says. It is interesting that the liver was where Kociba at Dow recognized TCDD as a carcinogen. A simple test for the presence of cytochrome P4501A.2 is being worked on. According to Birnbaum, the hydroxylase acts at a specific site on the caffeine molecu!.e. By labeling caffeine with carbon-13 at that site and administering it to people, the 1A2 cytochrome can be ctetected because13C-labeled carbon dioxide will be exhaled. "We are doing this now in experimental animaD.s," Birnbaum says. EPA's lab will be assessing the endpoints for 11 differ- ent responses in the same animal. The idea is to see at what da~e responses occur. Birnbaum says the data will be used to develop a risk model that might tell EPA just where humans fit into the overall scheme of toxicity of TCDD and dioxinlike compounds. If one assumes that people are walking around with about 100 ppt of TCDD toxicity equivalents, the assessment would be a guide- line as to what response might come from that. "We mav see that 100 ppt is orders of magnitude below the toxico- logical in.Election point," Birnbaum says, "and therefore not of great concern. On the other hand, if the current exposure levels put us right near that inflection point, then any additional exposure would be undesirable." She adds that EPA particularly needs to look at the issue of sensitive populations, such as subsistence fishermen and nursing infants, that might receive doses 10 to 20 times higher than the overall population. A great deal has been made about the issue of there being so ;peat a species difference in response to TCDD, Birnbaum says. However, for most toxicological end- points, such as death, researchers can find a species out- laver. In thi.s case it may be guinea pigs, which die at ex- posures off less than 1 µg per kg body weight. For the re- lated hamster, the dose at which half die may reach as high as 50U0 µg per kg body weight. "But most species cluster their sensitivity somewhere within a 10-fold range," shesays. "If you take other endpoints for TCDD, say developmental toxicity, the dose that will kill the developing fetus is essentially w ithin an order of magni- tude in the guinea pig, hamster, rat, and mouse. If you look at en~,me induction, the dose that causes a re- sponse in these animals is essentially the same. So lethal- ity has been sort of a red herring." Looking at the endpoint nformation on humans shows that they fall into the same range of sensitivities, Birnbaum says. "For enzyme induction and chloracne, humans respond similarly to experimental animals. In in-vitro expenments, the concentrations of TCDD that result in cleft palate formation in the rat and in the hu- man are essentially the same. For cancer, the recent study by Fingerhut is at least compatible with tl- pothesis that the blood levels in the group that 1, creased cancer were similar to the blood levels in the rats that developed cancer in the Kociba study." Birnbaum thinks there is no reason to believe that people are different from animals in their response to TCDD. "I think the Seveso data tell us that, in terms of lethality, we are not guinea pigs. At the highest doses received by residents near Seveso, if they had been guinea pigs, there would have been some deaths. But we never reached the levels of TCDD that would have killed rats or mice or monkeys or dogs 6r rabbits or minks or anything else." EPA's review will bring up to date the scientific rea- soning for TCDD toxicity. By merging the human epi- demiology data, the animal toxicity information, and the molecular biology, a better level of understanding and maybe a firmer basis for regulation of this conten- tious chemical will emerge. Silbergeld and Birnbaum both think the regulatory number for TCDD may not change much. Even though the response level for cancer is high, the response level for immunotoxicity may be very low, and the current safe intake calculation of 0.006 picogram per kg body weight per day may be in the right range. CDC's Houk has commented that the intake number seems too low. Given the history of TCDD, it is likely that politics and emotion will have as much say in the end as does sci- ence. Politics and emotion have a lot to do with the pt lic's fear of dioxin. Those embroiled in the public con- troversy have diverse views on why TCDD remains so persistently in the forefront of people's concerns. Stan- ford's Whitlock believes it is because the compound has become synonymous with horrible scenes of birth de- fects and cancer. "It has become a sort of prototype for certain groups who are concerned about the environ- ment; " he says. Several people place the focus on the Vietnam veter- ans. The Air Force's Wolfe, for one, thinks, "It was so closely related to the Vietnam experience and the ill treatment a lot of the returning folks got." But he also says, "It is sort of a flagship issue of all environmental problems." Houk says attention is rapt because of those who in- sist that TCDD be labeled the most toxic substance known to man. Joseph Walker from the Chlorine Insti- tute concurs on that point, adding that it also may be because "dioxin" looks and sounds like "toxin." But uncertainty may be the biggest reason people's concerns haven't been eased by science. NIOSH's Fin- gerhut speculates that people have never felt reassured by the information coming out. "It is hard to get an- swers for a problem like this, and they are long in com- ing," she says. Banbury conference organizer Gallo agrees. "I think the scare comes from some people say- ing there is no problem and others saying this is the most heinous compound man has ever created." At Maryland, Silbergeld blames the uncertainty on the government. "Primarily it's the government's fault that they can't come to a decision on TCDD regulation and stick to it." 1) 14 August 12 185, 1 C&EN