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PCBs (Polychlorinated Biphenyls) Structure-Activity Relationships ironmental Research Lab.-Duluth, MN (U.S.) Env ptkfio**t-U, m 9HM PB83-247486 Aug 83 U.S. DEPARTMENT OF COMMERCE National Technical Information Service NTIS \ RcC?.lV0 EX P-2733 Page 1 of 26 ! PCB-ARCH-EXT0378171 o . EPA-600/D-83-096 August 1983 ?33 3 -2 7!) 36 PCBs: STRUCTURE-ACTIVITY RELATIONSHIPS S. SAFE1, A. PARKINSON2, l. ROBERTSON1, T. SAWYER1 S. 3AN0IERA1, L. SAFE1, M.A. CAMPBELL3 AND M. MULLIN4' department of Physiology and Pharmacology, 'College of Veterinary Medicine, Texas A&M University, College Station, TX 77843 2 Department of Biochemistry and Drug Metabolism, Hoffman La Roche Inc., Nutley, NJ 07110 3 Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry, University of Guelph, Guelph, Ontario NIG 2W1 4E.P.A., Grosse lie Laboratory, 9311 Groh Roa- d, Grosse He, Michigan 48138 ENVIRONMENTAL RESEARCH LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY DULUTH, MN 55804 REPRODUCED 8Y NATIONAL TECHNICAL INFORMATION SERVICE . U 3. DEPARTMENT OF-COMMERCE SPRINGFIE1D. VA. 22161 EX P-2733 Page 2 of 26 r PCB-ARCH-EXT0378172 TECHNICAL REPORT OAIA . [Pleat read Inurucrioru cm tht revert* iw/ert cor>oUd^xj 1. REPORT NO. 3. EPA-600/D-83-096 X RECt!INT'S ACCESSION NO. roj 3 247 S8 6 4. TITLE ANO SUBTITLE PCBs: Structure-Activity Relationships S. REPORT DATE August 1983 . PERFORMING ORGANIZATION COO* AUTHOfllS) S. Safe, A. Parkinson, L. Robertson, T. Sawyer, S. Bandiera, L. Safe, M.A. Campbell, and M. Mullin* . PERPORMING ORGANIZATION NAM* ANO ADORES* *U.S. Environmental Protection Agency Large Lakes Research Station 9311 Groh- Road Crosse lie, Michigan 48138 Environmental Research Laboratory Office of Research and Development U.S. Environmental Protection Agency Duluth, m 55804 *. PERFORMING ORGANIZATION REPORT NO. 10. PROGRAM ELEMENT NO. TTT con tract/g^Ant no. IX TYPE OP REPORT ANO P6RIOO COVERED 14. SPONSORING AGENCY COOE EPA/600/03 IX SU>*L6M6NT*y NOTES Presented at: Recent Advances in Exposure, Health and Environmental Effects Studies of PCBs; Bethesda, Maryland, May 12-13, 1982. IX ABSTRACT - This report summarizes research on the chemical and toxicological characterization of PCB's. Results on the synthesis and characterizatin of all 209 PCB's, and subsequent identification of individual PCB components in commercial mixtures and environmental samples are reported. This was essential for research relating the toxicity and biologic effects of commercial mixtures to chemical structure. The results of structure-activity research with the various congeners on several biological systems are also reported. . 7. KIY WORD* ANO DOCUMENT ANALYSIS x descriptors b.lOENTIPIERSVOPEN ENDED TERMS c. COSATI Field/Croup 1*. DISTRIBUTION statement 19. SECURITY CLASS fTTUt Rtportf UNCLASSIFIED . 20. SECURITY CLASS (Thi* p*ft/ UNCLASSIFIED MvlOUl COITION II OIIOLSTC 21. NO. OP PAGES 25 23. PRICB EX P-2733 Page 3 of 26 PCB-ARCH-EXT0378173 NOTICE This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorse ment or recommendation for use. 11 EX P-2733 Page 4 of 26 PCB-ARCH-EXT0378174 INTRODUCTION Polychlorinated biphenyls (PCBs) are readily synthesized by the direct chlorination of biphenyl using iron salts and/or iodine as the catalyst. The degree of chlorination of the resulting mixture is con trolled by the relative amount of chlorine used in the reaction and most commercial products are graded and sold by their weight per cent chlorine content [1]. PCBs have been widely used in industry as heat transfer fluids, hydraulic fluids, solvent extenders, flame retardants, organic diluents and dielectric fluids. The unusual industrial versatility of PCBs is directly related to their physical properties which include:, a) resistance to acids and bases, b) compatibility with organic mater ials, c) resistance to oxidation and reduction, d) excellent electrical insulating properties, e) thermal stability and f) non-flanmiahi1ity. Unfortunately, these physical properties coupled with their widespread use, relatively low acute toxicity and improper disposal has resulted in the contamination by PCBs of every component of the global ecosystem [1-5]. Moreover, the lipophilic nature and persistence of PCBs also con tributes to their high bioaccumulation potential and their biomagnifica tion in higher trophic levels of the food chain [6]. PCBs residues are routinely detected in fish, wildlife and human adipose tissue, blood and breast milk. COMMON ANALYTICAL AND TOXICOLOGICAL PROBLEMS The analysis of PCB residues in environmental samples present num erous problems which include the following: l EX P-2733 Page 5 of 26 PCB-ARCH-EXT0378175 1. PCB formulations and environmental isolates are highly complex mixtures of isomers and congeners and 2. the composition of PCBs obtained from diverse environmental matrices are highly variable due to the different rates of biodegradation, vaporization, photodegradation and chemical degradation of the individual PCB isomers and congeners [6-8]. There are 209 possible PC3 isomers and congeners and most of the commercial mixtures and environmental samples exhibit a multitude ofpeaks and this is particularly evident in the capillary gas chromato gram of the commercial PCB, Aroclor 1260. It has been common practice to quantitate environmental PCBs by comparing their packed column gas chromatograms with the patterns exhibited by known amounts of individual commercial PCBs or mixtures of these formulations [9,10]. This method relies on pattern matching using specific peaks for quantitation. It is clear that if the gas chromatograms of the environmental PCB residues cannot be "matched" with an appropriate cocktail containing known amounts of the commercial formulations then quantitation is not possible. Com parable analytical problems have been encountered in the analysis of other complex halogenated aromatic pollutants, and polynuclear aromatic hydrocarbons [11], These problems experienced in PCB analysis by gas chromatography (GC) can be resolved in the following way: 1) the samples must be analyzed by high resolution capillary gas chromatography to ensure the maximum separation of the isomers and congeners, 2) all 209 PCB isomers and congeners must be available as analytical standards for the identification and quantitation of the indi vidual components in the analyte. 2 EX P-2733 Page 6 of 26 PCB-ARCH-EXT0378176 In collaboration with Dr. M. Mull in and several coworkers, consid erable progress has been made on the synthesis and characterization of all 209 PCBs (Table 1) and this has resulted in the positive identifi cation of the individual PCB components present in commercial mixtures and in environmental samples. Based on the numbering scheme proposed by BalIschmitter and Zell [13], Figure 1 illustrates the identification of the individual PCBs in Aroclor 1260 using the synthetic PCB standards [12]. ' 4 - Table 1. Progress in the Synthesis of PCB Isomers and Congeners Cl Groups 1 2 3 4 5 6 7 8 9 10 Total Mo. of Isomers 3 12 24 42 46 42 24 12 3 1 209 No. Synthesized or Available 3 9 18 40 42 40 24 12 3 1 192 Most reviews discussing the biologic and toxic effects of commercial PC3s note that the relative potencies of these formulations depends on their degree of chlorination and also on the species being used in the study [14,15], The results clearly suggest that since the toxicities of the mixtures are variable, then the activities of the individual com ponents of these mixtures may also be different. Research on the com parative biologic and toxic effects of PCBs has clearly illustrated the marked differences in the comparative toxicities of PCB congeners as noted below. * T) 2,3',4,4',5-Pentachlorobiphenyl is highly embryotoxic to chickens whereas 2,2' ,5,5'-tetra and 2,2',4,4*,5,5'-hexachlorobiphenyl are relatively non-toxic [16]. 3 EX P-2733 Page 7 of 26 PCB-ARCH-EXT0378177 2) 3,3',4,4'-Tetrachlorobiphenyl is highly toxic to Rhesus macaques whereas the 2,2',5,5'-isomer elicits no clinical effects [17]. 3) The toxic lesions caused by 3,3',4,4',5,5'-hexachlorobiphenyl in male mice were much more severe than those observed after administration of the 2,2' ,4,4' ,6,6'-, 2,2',4,4',5,5'- and 2,2' ,3,3',6,6'-hexachlorobiphenyls [18]. 4) A comparison of the toxicity of 2,3,3' ,4,4'-penta- and 2,3' ,4,4'tetrachlorobiphenyl showed the former congener to be highly active and the latter compound to be inactive [19]. ' 5) Chronic administration of 2,2',3,3',4,4'- and 2,2',3,4,4',5'hexachlorobiphenyl caused an accumulation of liver porphyrins whereas administration of the 2,2',4,4',5,5'-hexachlorobiphenyl had no effect on hepatic porphyrin levels [20]. 6) 3,3',4,4',5- and 2,3' ,4;,4' ,5-Pentachlorobiphenyl and 2,3,3',4,4',5- hexachlorobiphenyl cause thymic atrophy in rats whereas the 2,2',3,3',4,4',5- and 2,2],3,4,4',5,5'-heptachlorobiphenyls were not toxic [21]. It is evident that subtle changes in the structure of PCBs can re sult in marked differences in toxicities. Thus, an assessment of the potential long term effects of PCBs on biological systems will depend on the concentration of the individual toxic congeners which are pres ent and which persist in the animal species. This necessitates not only a high resolution quantitative and qualitative analysis of PCB residues, . but prior knowledge concerning the potential or actual toxicities of the individual PCBs. . 4 o EX P-2733 Page 8 of 26 PCB-ARCH-EXT0378178 PCBs AS INDUCERS OF HEPATIC MICROSOMAL QRUG-META80LIZING ENZYMES One of the most characteristic biologic effects of the commercial PCBs is their induction of numerous hepatic microsomal drug-metabolizing enzymes in rodents and particularly the cytochrome P-450-dependent mono oxygenases [14,22-25]. Administration of these mixtures results in increases in microsomal aniline hydroxylase, benzo[a]pyrene hydroxylase, biphenyl hydroxylases, halobiphenyl hydroxylases as well as numerous N- and 0-dealkylases. The potent induction of these cytochrome P-450 dependent monooxygenases by Aroclor 1254 has resulted in the widespread use of PCB-induced microsomes as the source of metabolic activation for chemicals in the Ames test for their bacterial mutagenicity [26]. Inducers of microsomal monooxygenases are classically subdivided into two main categories [27-29]. One class is typified by phenobar- bitone (PB) and the other by 3-methylcholanthrene (MC). P3-type inducers enhance microsomal N-demethylase (2-3-fold), NADPH cytochrome P-450 reduc tase (2-3-fold) biphenyl-4-hydroxylase (2-4-fold) and aldrin epoxidase (3-5-fold) whereas MC-type inducers enhance microsomal benzo[a]pyrene hydroxylase (8-12-fold), 4-chlorobiphenyl hydroxylase (8-10-fold), ethoxy- resorufin Q-deethylase (up to 30-fold) and biphenyl 2-hydroxylase (8-12 fold). The preferential induction of specific monooxygenases by MC- and PB-type inducers coupled with the different electrophoretic and spectral properties of their induced microsomal enzyme preparations clearly dif ferentiates between the two classes of inducers. The enzyme induction properties of Aroclor 1254 and other cormerciai PCBs do not resemble that of either PB or MC but are similar to the pattern produced after co administration of PB + MC [22-24]. More recent studies have shown that Aroclor 1254 induces cytochromes P-450b and.P-450c which are also the major isozymes induced by PB and MC respectively [25]. The mixed-type 3 EX P-2733 Page 9 of 26 PCB-ARCH-EXT0378179 induction activity of Aroclor 1254 was presumed to be due to the indi vidual PB- and MC-type inducers present in this mixture. PCBs AS MICROSOMAL MONOOXYGENASE ENZYME INDUCERS: EFFECTS OF STRUCTURE ON ACTIVITY PB-Type Inducers Initial studies suggested that PCBs which are PB-type inducers must contain at least two para and two meta substituents; any further substi tution changes only the quantitative nature of the induction activity [30.31]. There is ample evidence for these correlation and some of the most active PB-type inducers are 2,2' ,4,4'-tetra-, 2 ,2',4,4',5,5'hexa-and 2,2',3,3` ,4,4'-hexachlorobiphenyl. However it has subsequently been shown that PC8 congeners which contain one or no para substituents, are also PB-type inducers [32]. .Some of these compounds include 2,2',3,3',5,5' hexachlorobiphenyl, 3,3'-dichlorobipher,yl, 2,3,3',5,5',6-hexachlorobiphenyl and 2,3,3',4',5,6-hexachlorobiphenyl. The requirement for at least two ortho chloro substituents conflicts with the reported activity of 4,4'dichlorobiphenyl and 2,3' ,4,4`-tetrachlorobiphenyl as PB-type inducers [19.32]. Table 2 illustrates the structurally diverse group of PCBs which are classified as PB-type inducers and it is evident that there are no apparent structure-activity rules for PCBs which enhance this pattern of enZyme activity. MC-Type Inducers In 1977, two groups [30,31] published papers outlining structureactivity rules for PCBs as inducers of cytochrome P-448 dependent mono- EX P-2733 Page 10 of 26 PCB-ARCH-EXT0378180 Table II. PCBs as PB-Type Inducers Congener Number of Chloro Substituent Ortho Meta Para 4,4'-dichlorobiphenyl 0 0 0 2,3',4,4'-tetrachlorobiphenyl 1 1 2 2,2' ,4,4'-tetrachlorobiphenyl 2 0 2 3,3`-dichlorobiphenyl 0 2 0 2,3,3' ,4,5,5'-hexachlorobiphenyl 1 4 1 Reference 30, 31 19 30, 31, 36 31 36 4 oxygenases (or aryl hydrocarbon hydroxylase, AHH). Their data indicated that PCBs which exhibit this type of induction activity must be substi tuted in both para positions and at least one meta position on both phenyl rings and not contain any ortho-chloro substituents. The three compounds defined by these rules, namely 3,3' ,4,4'-tetra-, 3,3',4,4',5-penta- and 3,3' ,4,4',5,S'-hexachlorobiphenyl are all potent AHH inducers and are approximate isostereomers of 2,3,7,8-tetrachlorodibenzo--dioxin (TCDO), the most toxic halogenated aromatic chemical and most potent inducer of microsomal AHH [33].. It was apparent that the proposed structure-activity rules did not explain the mixed-type induction activity of the commercial mixtures since the three AHH inducers are only trace components of the active commercial PC3s [3,13,33]. For this reason, the effects of structure on the activity of PCBs as AHH inducers were reevaluated using a series of highly purified synthetic PCS congeners [32,34-39]. 7 EX P-2733 Page 11 of 26 PCB-ARCH-EXT0378181 Requirements for Para Substituents The requirements for para substituents were tested using a series of PCS congeners with one, two and zero para substituents and in all cases PCBs which were AHH inducers all contained two para-chloro groups. (Figure 2). Requirements for Meta Substituents The requirements for meta substituents were evaluated by, testing all the possible meta-substituted PCBs derived from 4,4'-dichlorobiphenyl, a PCS which contains the required two para-chloro groups. Figure 2 de notes the five possible compounds in this series and only the 3,3',4,4'tetra-, 3,4,4' ,5-tetra, 3,3*,4,4',5-penta- and 3,3',4,4',5.5'-hexachlorobiphenyls were AHH inducers. Thus one new PCS congener, namely 3,4,4',5tetrachlorobiphenyl is added to the original list of three AHH inducers and it is apparent that two meta substituents are required for PCBs which induce AHH, however they can be positioned on both or the same phenyl ring. Requirements for Ortho Substituents A logical approach for evaluating the effects of ortho-chloro substi tuents on the activity of PCBs as AHH inducers is to synthesize and test all the possible mono- and diortho-substituted analogs of the four MC-type inducers. A summary of these compounds is given in Figure 3. All the mono-ortho substituted analogs were mixed-type inducers in the immature male Wistar rat and were inducers of AHH in rat hepatoma H-4-II-E cells in culture. Moreover many of these compounds, including 2,3,3',4,4'- penta-, 2,3',4,4',5-penta-, 2,3,3',4,4',5-hexa- and 2,3,3',4,4',5 ,5'- EX P-2733 Page 12 of 26 PCB-ARCH-EXT0378182 heptachlorobiphenyl have been identified as components of the commer cial PCBs [3,13,33]. The effects of two ortho chloro substituents might be expected to have a more pronounced effect on the population of the planar conformers ' and, hence, on their activity as MC-type inducers. Previous reports in the literature suggested that PCBs substituted in at least one meta and para position on each phenyl ring and at two ortho positions were strictly PB-type inducers as evidenced by the activity of the 2,2',3,3',4,4'- and 2,2' ,4,4` ,5,5'-hexachlorobiphenyl isomers [30,31]. Contradictory re ports have suggested that these isomers also possess MC-type activity [20,40], A subsequent reinvestigation of the activity of 2,2',4,4',5,5'- hexachlorobiphenyl as a microsomal enzyme inducer has shown that the MC- type activity of a 99: pure commercial sample (prepared by the Ullmann coupling method) was due to contamination with the highly active 2,3,7,8- tetrachlorodibenzofuran (TCDF) [41]. In attempt to resolve conflicting reports in the literature', the ef fects of 2,2' ,3,3',4,4`-hexa-, 2,2',4,4',5,5'-hexa- and 2,2',3',4,4',5- hexachlorobiphenyl on the hepatic microsomal drug-metabolizing enzymes were evaluated in the immature male rat [37]. By comparison with the effects of the classical enzyme inducers, PB and MC, 2,2',4,4',5,5'-hexa chlorobiphenyl was classified as a pure PB-type inducer. In contrast, 2,2' <3,3' ,4,4'-hexachlorobiphenyl, irrespective of its synthetic route, exhibited PB-type and weak MC-type characteristics; the most prominent feature of the latter being a 7-fold increase in 4-chlorobiphenyl hydroxy lase activity. 2,2',3',4,4',5-Hexachlorobiphenyl also resembled a mixed (PB + MC)-type inducer although its MC-type characteristics were more pronounced that those of 2,2',3,3',4,4'-hexachlorobipheny 1. Since these three hexachlorobipheny1s are di-ortho substituted derivatives of the pure EX P-2733 q Page 13 of 26 PCB-ARCH-EXT0378183 MC-type inducer, 3,3' ,4,4'-tetrachlorobiphenyl, it is clear that the presence of two ortho chlorines does not necessarily abolish MC-type character. A detailed study of all the di-ortho substituted analogs of the four AHH inducers indicated that, in addition to the two PCB isomers noted above, 2,3,4,4' ,5,6-hexa-, 2,3,3',4,4',6-hexa- and 2,2',3,3',4 ,4',5-heptachlorobiphenyl were also mixed-type inducers [38]. Therefore the PCBs which induce MC-type activity must be substi tuted at both para positions, at least two meta positions (but not aeces- sarily on different phenyl rings) and can also contain one ortho substi tuent; the addition of a second ortho chloro substituent to the nucleus of an MC-type inducer does not necessarily eliminate the qualitative aspects of this activity if one of the ortho substituents is part of a 2,3,4-trichlorophenyl substitution pattern. PCSs Which Do Not Induce Microsomal Monooxygenuses It has also been shown that several PCBs containing one to three chlorine atoms or biphenyls chlorinated on only one of the phenyl rings are weak or inactive as inducer of cytochrome P-450 dependent monooxygen ases [30,31]. Their lack of activity may be due to their more rapid .rate of metabolism and clearance. Some of the more highly chlorinated octa-decachlorobiphenyls are also inactive [30,31,34]. Another group of PCBs which are.inactive as AHH inducers contain 3,4 or 3,4,5 substitution on one phenyl ring with a 2,4,6- or 2,3,5-trichloro substitution pattern on the second ring [36,42,43]. These congeners do accumulate in the liver and contain one or two ortho-chloro groups, however, for some reason they are inactive as a microsomal monooxygenase inducer and are relatively non-toxic. The rationale for these results is not apparent. 10 EX P-2733 Page 14 of 26 PCB-ARCH-EXT0378184 PCBs: CORRELATION BETWEEN AHH INDUCTION, Ah RECEPTOR BINDING AND TOXICITY Poland and coworkers have stated "The potency of halogenated aromatic hydrocarbons to induce hepatic AKH activity correlates very closely to their toxic potency" [44] and "that the .toxicity of these chlorinated aromatic hydrocarbons is mediated through the receptor; that is the initial event in the toxic action is the stereo-specific recognition and binding of them by the cytosolic binding species" [33]. The evi dence for this hypothesis is based on the biologic and toxic effects of several congeneric polychlorinated dibenzo--dioxins (PCDDs) and halogenated aromatic compounds which are approximate isostereomers of 2,3,7,8-TCOO [33], The results which support the proposed correlations are noted below: . 1) there is an excellent correlation between the toxic potency of PCDD congeners, their AHH induction activity and affinity for the AH cytosolic receptor protein (33, for example 2,3,7,8- TCDD is highly toxic, a potent AHH inducer and avidly binds the Ah receptor protein; 2,7,-dichlorodibenzo-^-dioxin is relatively non-toxic, does not bind to the receptor protein or induce micro somal AHH), " 2) 2,3,7,8-TCDD and related approximate isostereomers are rela tively toxic to the genetically inbred C578L/6J "responsive mice" and non-toxic to the non-responsive DBA/2J mice [33,42], 3) 2,3,7,8-TCDD and approximate isostereomers induce AHH in the responsive mice however only 2,3,7,8-TCDD is active (at higher concentrations) in the DBA/2J mice [33,44-46]. 11 ' EX P-2733 Page 15 of 26 PC B-ARCH-EXT0378185 PC3s are an ideal group of-halogenated aromatic chemicals to test and confirm the correlations proposed by Poland and coworkers. Table 3 illustrates the relative AHH inducing activities of 3,3' ,4,41-tetra-, 3,4,4',5-tetra-, 3,3',4,4' ,5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl and their mono-ortho chloro analogs. The results illustrate that with few exceptions there is an excellent correlation between the relative potencies of the individual PCB congeners as AHH inducers in Wistar rats and rat hepatoma H-4-II-E cells and their avidity for the Ah cytosolic receptor [47]. Although the toxicities of these mono-ortho chlorinated congeners has not been systematically studied there are reports in the literature [16,19,21] which confirm the comparatively high toxicity of the 2,3,3',4,4'-penta-, 2,3',4,4',5-penta- and 2,3,3',4\4',5-hexachlorobiphenyl. Current research in my laboratory [48,49] with genetically Table III. PCBs - Effects of Structure on Activity PCB Congeners (number) Relative Per Cent Activity AHH Induction MC-Type Inducers (3) Cell Cultures 100-1 Rats f *+ Mixed-Type Inducers (8) 0.3-2.4x10-5 ++ PB-Type Inducers (4) inactive inactive Non-Inducers (3) 2,3,7,8-TCDD inactive 400 inactive +++++ Receptor Binding 100-35 6-1.5 v 0.3 < 0.3 2,500 inbred mice indicates that administration of the mixed-type PCB inducers to the responsive C57BL/6J mice results in AHH induction and thymic atrophy whereas these compounds do not induce AHH nor are they toxic in the nonresponsive 0BA/2J mice. 12 EX P-2733 Page 16 of 26 PCB-ARCH-EXT0378186 The results obtained for the PCBs complement the data reported for several PCOO congeners and support the correlations noted by Poland and coworkers [33,34]. Moreover, it is also apparent that PCB congeners which induce AHH activity are toxic and the residue levels of these specific congeners in fish wildlife and exposed humans may be important indicators of potential short and long term toxicity. PCBs IN HUMANS: PRELIMINARY STUDIES Figure 3 summarizes al1 the PCBs expected to exhibit MC-type activity based on the above guidelines and a number of these isomers and congeners have been identified in commercial PCBs and in humans (blood, adipose tissue and breast milk) [50-52]. As noted in Table IV there is relatively high concentration of MC-type inducers which preferential1y bioconcentrate in human breast milk. A recent study in our laboratory has shown that the dose effecting half-maximal (EO50) induction of AHH activity for a. reconstituted breast milk PCB sample was 12 tmiol-kg"1 whereas the ED5Q for Kanechlor 500, a commercial PCB mixture, was 87 ;>mol-kg"l. Thus the increased biological potency of breast milk PCBs reflects the preferential bioconcentration of 2,3',4,4',5-penta-, 2,3,3',4,4'-penta-, 2,3,3',4,4' ,5hexa- and 2,2',3',4,4',5-hexachlorobiphenyl which are mixed-type inducers and elicit various toxic responses in the rat, chicken and responsive C57BL/6J mice [16,19,21,48,49]. \ 13 EX P-2733 Page 17 of 26 PCB-ARCH-EXT0378187 Table IV. Breast Milk and Reconstituted Breast Milk PCBs [50-53] PCB Structure GC Purity () PCB Concentration in Japanese Breast Milk (;,) PCB Concentratior in Reconstituted Breast Milk Mixture {%) 2,4,4'-Trichlorobiphenyl 2,2' ,5,5'-Tetrachlorobiphenyl 2,4,4' ,5-Tetrachlorobiphenyl 2,2' ,4,5,5'-Pentachlorobiphenyl 2,3' ,4,4' ,5-Pentachlorobiphenyl 2,2' ,3,4* ,5,5'-Hexachlorobiphenyl 2,3,3',4,4'-Pentachlorobiphenyl 2,2' ,4,4' ,5,5'-Hexachlorobiphenyl 2,2' ,3,4,4' ,5'-Hexachlorobiphenyl 2,2' ,3,4' ,5,5',6-Heptachlorobiphenyl 2.2',3,4,4',5` ,6-Heptachlorcbiphenyl 2,3,3' ,4,4' ,5-Hexacnlorobiphenyl 2,2' ,3,4,4',5,5'-Heptachlorobiphenyl 2,2* ,3,3',4,4',5-Heptachlorobipnenyl Total 98.5 99.0 99.0 93.5 99.0 99.0 99.0 99.0 99.0 98.5 99.0 99.0 90.0 98.5 3.4 4- 2.2 2.0 4 1.3 19.1 + 2.6 2.8 + 0.9 11.8 + 1.2 2.3 + 0.3 3.5 + 0.5 15.5 + 0.4 15.3 + 0.7 . 3-2 + 0.6 1.6 + 0.3 2.1 + 0.7 5.1 + 0.7 2.3 + 0.3 95 .5 9.2 2.2 20.1 3.0 12.8 - 3.9 16.3 16.9 3.3 1.7 2.2 5.7 2.3 99.9 ACKNOWLEDGEMENTS The financial assistance of The U.S. Environmental Protection Agency, Health and Welfare Canada, the National Institutes of Health (1-R01-ES02798- 01), the Center for Comparative Medicine, the Natural Sciences and Engineer ing Research Council of Canada and the Texas Agricultural Experiment Station (Grant Number 6376) is gratefully acknowledged. . 14 EX P-2733 Page 18 of 26 PCB-ARCH-EXT0378188 Ln Figure 1. High resolution glass capillary gas chromatoqram of Aroclor 1260 EX P-2733 Page 19 of 26 PCB-ARCH-EXT0378189 I o Figure 2. Effects of para and meta substituents on the activity of PCBs as AHH inducers; active ( + ), inactive (-). o EX P-2733 Page 2_0 of 26 O' ci 2 3 3'4 4' 2 3'4 4'5 2 2'4 4' 5 5' 2 2 '3 3 ' 4 4 ' 2 2'3 4 4*5' 2 33'44'6 ci 2 3 3 '4 4 ' 5 2 3 '4 4 ' 5 5 ' 2 3 3' 4 4*5 ' 2 2'3 3 '4 4 ' 5 2 2'3 4 4' 5 5' 2 3 3'4 4' 5 6 2 3 3'4 4'5' 6 c, 2 3 3'4 4 '5 5' 2 2' 3 3 ' 4 4 ' 5 5' 2 3 3'4 4'5 5' 6 2' 3 4 4'5 2 344' 5 2 3 44'5 6 2 2' 3 4 4' 5 23'44'5'6 Figure 3. Mono and di-ortho substituted analogs of 3,3` ,4 ,4'-tetra-, 3,4,41,5-tetra-, 3,3`,4,4',5-pentar and 3,3`,4,4*,5,5'hexachlorobiphenyl. EX P-2733 Page 21 of 26 PCB-ARCH-EXT0378191 t REFERENCES 1. Hutzinger, 0., S. Safe and V. Zitko. "The Chemistry of PCB's", (CRC Press, Inc., Ohio, 1974). .2 Landrigan, P.J. "General Population Exposure to Environmental Con centrations of Halogenated 8iphenyls" in Halogenated Biphenyls, Terphenyls, Naphthalenes, Oibenzodioxins and Related Products, (R.O. Kimbrough, ed. , Elsevier/North Holland, Amsterdam, 1980), p. 267. 3. Jensen, S. and G. Sundstrom. "Structures and Levels of most Chloro- biphenyls in Two Technical PCS Products and in Human Adipose Tissue", Ambio, 3: 70-76 (1974). 4. Wassermann, M. , 0. Wassermann, S. Cucos and H.J. Miller.* "World PC8s Map: Storage and Effects in May and His Biologic Environment in the 1970`s", Ann. N.Y, Acad. Sci. 320: 69-124 (1979). 5. Fujiwara, K. "Environmental and Food Contamination With PCBs in Japan", Sci. Total. Environ. 4: 219-247 (1975). 6. Safe, S. "Metabolism, Uptake, Storage and Bioaccumulation", In: Halogenated Biphenyls, Naphthalenes, Oibenzodioxins and Related Products (ed. R. Kimbrough ~ Elsevier/North-Holland, 1980), p. 77. 7. 3a11schmiter, K., M. Zell and H.J. Neu. "Persistence of PCBs in the Ecosphere: Will Some PCB-Components Never Deqrade" Chemosphere, 7: 173-1 76 (1978).' 8. Harvey,G.R. and W.G. Steinhauer. "Atmospheric Transport of Polychlorobiphenyls to the North Atlantic" Atmos. Environ. 8: 777-782 (1974). 9. Sawyer, L.O. "Quantitation of Polychlorinated 3ipheny1 Residues by Electron Capture Gas-Liquid Chromatography: Collaborat've Study, J. Assoc. Offic. Anal. Chem. 61: 282-291 (1978). 10. Albro, P.W., S.T. Corbett and J.L. Schroeder. "Quantitative Charac terization of Polychlorinated Biphenyl Mixtures (Aroclors 1248, 1254 and 1260) by Gas Chromatography Using Capillary Columns, J. Chrom. 205: 103-111 (1981). ' ~ .11 "Polychlorinated 0ibenzo--dioxins: Limitations to the Current Ana lytical Techniques", National Research Council Canada, Publication NRCC 18576, (1981). .12 Mullin, M., G. Sawka, L. Safe, S. McCrindle and S. Safe. "Synthesis of Octa- and Nonachlorobiphenyl Isomers and Congeners and Their Quantita tion in Commercial Polychlorinated Biphenyls and Identification in Human Breast Milk" J. Anal. Toxicol., 5: 138-142 (1981). 18 EX P-2733 Page 22 of 26 PCB-ARCH-EXT0378192 13. Ballschmiter, K. and M. Zell. "Analysis of Polychlorinated Biphenyls (PCB) by Glass Capillary Gas Chromatography. Composition of Technical Aroclor- and Clophen-PCB Mixtures", Fresenius Z. Anal. Chem., 302: 20 31 (1980). ------------------- 14. Ecobichon, O.J. and A.M. Comeau. "Comparative Effects of Commercial Aroclors on Rat Liver Enzyme Activities", Chein.-Biol. Interact.. 9: 241-350 (1974). ' ------------------------------------ 15. Kimbrough, R.O. "The Toxicity of Polychlorinated Polycyclic Compounds and Related Chemicals", CRC Crit. Rev. Toxicol. 2: 445-448 (1974). 16. Ax, R.L. and L.G. Hansen. "Effects of Purified PCB Analogs on Chicken Reproduction", Poultry Sci., 54: 895-900 (1975). 17. McNulty, W.P., G.M. Becker and H.T. Corey. "Chronic Toxicity of 3,3',4,4'-Tetrachlorobiphenyls in Rhesus Macaques", Toxicol. Appl. Pharmacol. 56: 182-190 (1980). .......... ~~ " " 18. Biocca, M. , B.N. Gupta, K. Chue, J.D. McKinney and J.A. Moore. "Toxicity of Selected Hexachlorcbiphenyl Isomers in the Mouse" Toxicol. Appl, Pharmacol. 58: 461-474 (1981). 19. Yamamoto, H., H. Yoshimura, M. Fujita and T. Yamamoto. "Metabolic and Toxicologic Evaluation of 2,3,4,3' ,4-Pentachlor.obiphenyl in Rats and Mice", Chem. Pharm. Bull., 24: 2168-2174 (1976). ' 20.. Stonard, M.0. and J.B. Grieg. "Different Patterns of Hepatic Micro somal Enzyme Activity Produced by Administration of Pure Hexachloro- biphenyl Isomers and Kexachlorobenzene", Chein.-Biol. Interact., 15: 365-379 (1976). ~ 21. Yoshihara, S.., K. Kawano, H. Yoshimura, H. Kuroki and Y. Masuda. "Toxicological Assessment of Highly Chlorinated Biphenyl Congeners . Retained in the Yusho Patients", Chemosphere., 8: 531-538 (1979). 22. Alvares, A.P. and A. Kappas. "The Inducing Properties of Polychlor inated Biphenyls on Hepatic Monooxygenases". Clin. Pharmacol. Ther. 22: 809-816 (1977). " 23. Alvares, A.P., D.R. Bickers and A. Kappas. "Polychlorinated Biphenyls: A new Type of Inducer of Cytochrome P-448 in the Liver", Proc. Natl. Acad. Sci. (USA), 70: 1321-1325 (1973). 24. Alvares, A.P. and A. Kappas. "Heterogeneity of Cytochrome P-450`s Induced by Polychlorinated Biphenyls", J. Biol. Chem. , 252: 6373 6378 (1977). ' 19 EX P-2733 Page 23 of 26 PCB-ARCH-EXT0378193 25. Ryan, O.E., P.E. Thomas, 0. Korzeniowski and W. Levin. "Separation and Characterization of Highly Purified Forms of Liver Microsomal Cytochrome P-450 from Rats Treated with Polychlorinated Biphenyls, Phenobarbital, and 3-Methylcholanthrene", J. Biol. Chem. , 254: 1365 1374 (1979). 26. Hollstein, M. , J. McCann, F.A. Angelosanto and W.W. Nichols. "Short Term Tests.for Carcinogens and Mutagens" Mut. Res. 65: 133-226 ( 1979). 27. Conney, A.H. "Pharmacological Implications of Microsomal Enzyme Induction", Pharmacol. Rev., 19: 317-366 (1967). 23. Lu, A.Y.H., R. Kuntzman, and A.H. Conney. "The Liver Microsomal Hydroxycomponents", Front. Gastrpintest. Res., 2: 1 -31 ( 1976). 29. Slabek, N.E. and G.J. Mannering. "Evidence for a New P-450 Hemoprotein in Hepatic Microsomes from MethyIcholanthrene Treated Rats", Piochem. Bioohys. Res. Ccmmun., 24: 653-674 (1976). 30. Poland, A. and E. Glover. "Chlorinated Biphenyl Induction of Aryl Hydrocarbon Hydroxylase Activity: A Study of the Structure Activity Relationship", Mol. Pharmacol., 13: 924-938 (1977). 31. Goldstein, J.A., P. Hickman, H. Bergman, J.O. McKinney and M.P. Walker. "Separation of Pure Polychlorinated Biphenyl Isomers into Two Types of Inducers on the Basis of Induction of Cytochrome P-450, P-448", Chem.-Siol. Interact. , 1 7: 69-87 (1977). 32. Parkinson, A., R. Cockerline and S. Safe. "Polychlorinated Biphenyl Isomers and Congeners as Inducers of Both 3-Methylcholanthrene- and Phenobarbitone-Tvpe Microsomal Enzyme Activity", Chem, Biol. Interact. , 29: 277-239 (1980). 33. Poland, A., W.F. Greenlee and A.S. Kende. "Studies on the Mechanism of Toxicity of the Chlorinated Dibenzo--dioxins and Related Compounds", Ann, N.Y. Acad. Sci., 320: 214-230 (1979). 34. Parkinson, A. and S. Safe. "Aryl Hydrocarbon Hydroxylase Induction and its Relationship to the Toxicity of Halogenated Aryl Hydrocarbons", Toxicol. Environ. Chem., 4: 1-46 (1981). 35. Parkinson, A., R. Cockerline and S. Safe. "Induction of Both 3-Methyl cholanthrene- and Phenobarbitone-Type Microsomal Enzyme Activity by a Sinqle Polychlorinated Biphenyl Isomer", Biochem. Pharmacol., 29: 259-262 (1980). ' ......... 36. Parkinson, A., L. Robertson, L. Safe and S. Safe. "Polychlorinated Biphenyls as inducers of Hepatic Microsomal Enzymes: Structure-Activity Rules", Chem. Biol. Interact., 30: 271-285 (1981). o 20 EX P-2733 Page 24 of 26 PCB-ARCH-EXT0378194 I Parkinson, A., L. Robertson and S. Safe. "Hepatic Microsomal Enzyme Induction by 2,2',3,3',4,4'- and 2,2',3' ,4.4' ,5-Hexachlorobipheny1", Life Sciences, 27: 2333-2337 (1980). Parkinson, A., L. Robertson, L. Safe and S. Safe. "Polychlorinated Biphenyls as Inducers of Hepatic Microsomal Enzymes: Effects of Oi-Ortho Substitution", Chem. Biol. Interact. , 31: 1-12 (1981). Sawyer, T. and S. Safe. "PCS Isomers and Congeners Induction.of Aryl Hydrocarbon Hydroxylase and Ethoxyresorufin O-deethylase Activ ities in Rat Hepatoma Cells", Toxicol. Letters, in press. Alvares, A.P. "Stimulatory Effects of Polychlorinated Biphenyls (PCS) on Cytochromes P-450 and P-448 Mediated Microsomal Oxidations", In Microsomes and Qruq Oxidations (Ed: V. Ullrich et al., Pergamon Press, Oxford, 1977), p. 476-483. ' 4 Goldstein, J.A., J.R. Haas, P. Linko and D.J. Harvan. "2,3,7,8Tetrachlorodibenzo--di.oxin in Commercially Available 99* Pure Poly chlorinated Biphenyl Isomer Identified as the Inducer of Hepatic Cytochrome P-448 and Aryl Hydrocarbon Hydroxylase in the Rat", ' Drug Metab. Dispos., 6: 258-264 (1978). Goldstein, J.A., P. Linko, J.O. McKinney and P.W. Albro. "Marked in the Inductive Effects of Two Symmetrical Hexachlorobiphenyls and the Corresponding Unsymmetrical Isomer on Hepatic Monooxygenases", Siochem, Pharmacol., 30: 1008-1011 (1981).' 43. Parkinson, A. "Induction of Multiple Forms of Cytochrome P-450 by Polychlorinated Biphenyls", Ph.O. Thesis, University of Guelph, Guelph Ontario (1980). - ~ 44. Poland, E. and E. Glover. "2,3,7,8-Tetrachlorodibenzo-fj-dioxin: Segregation of Toxicity with' the Ah Locus, Mol. Pharmacol., 17: 86-94 (1980). ' ' " ~ ~~ ~ 45. Poland, A., E. Glover, and A.S. Kende, "Stereospecific, High Affinity Binding of 2,3,7,8-Tetrachlorodibenzo--dioxin by Hepatic Cytosol. Evidence that the Binding Species is Receptor for Induction of Aryl Hydrocarbon Hydroxylase", J. Biol. Chem., 251: 4936-4946 (1976). 46. Poland, A., E. Glover, J.R. Robinson and O.W. Nebert. "Genetic Expression of Aryl Hydroxylase Activity. Induction of Monooxygenase Activities and Cytochrome P-450 Formation by 2,3,7,8-Tetrachlorodibenzo--dioxin in Mice Genetically Nonresponsive to Other Aryl Hydrocarbons", J. Biol. Chem., 249: 5599-5606 (1974). 47. Bandiera, S., S. Safe and A.B. Okey. "Binding of Polychlorinated 8iphenyls Classified as Either PB-, MC- or Mixed-Type Inducers to the Cytosolic Ah Receptor Protein", Chem-Biol, Interact., in press. 21 EX P-2733 Page 25 of 26 _i PCB-ARCH-EXT0378195 1 I 43. Parkinson, A., L. Robertson, L. Uhl i g, M.A. Campbell and S. Safe. "2,3,4,4',5-Pentachlorobiphenyl: Differential Effects on the C57BL/6J and DBA/2J Mice", Biochem. Pharmacol., in press. 49. Safe, S., A. Parkinson, L. Robertson, S. Bandiera and M.A. Campbell, unpublished results. 50. Kuwabara, K., T. Yakushiji, I. Watanabe, S. Yoshida, K. Koyama and N. Kunita. "Increase in the Human Blood PCB Levels Promptly Follow ing Ingestion of Fish Containing PCBs", Bull. Envi ronm. Contam. Toxicol. 21: 453-462 ( 1979). ' " ............." * ~" 51. Yakushiji, T., I. Watanabe, K. Kuwabara, S. Yoshida, K. Koyama, I. Hara and N. Kunita. "Long-Term Studies of the Excretion of Polychlorinated Biphenyls (PCBs) Through the Milk of an Occupationally-xposed Worker", Arch. Environm. Contam, Toxicol., 7: 493-504 (1978). 4 52. Yakushiji, T., I. Watanabe, K. Kuwabara, S. Yoshida, K. Koyama and 11. Kunita. "Levels of Polychlorinated Biphenyls (PCBs) and Organo Chlorine Pesticides in Human Milk and Blood Collected in Osaka Prefecture from 1972 to 1977", Int. Arch. Qccup. Environm. Health, 43: 1-15 (1979). '..............' " ~ ' " 53. Parkinson, A., L.W. Robertson and S. Safe. "Reconstituted Breast Milk PCBs as Potent Inducers of Aryl Hydrocarbon Hydroxylase", Biochem. Siophvs. Res. Commun. 96: 382-889 (1930). / 22 EX P-2733 Page 26 of 26 PCB-ARCH-EXT0378196