Sexual dimorphism in hepatic microsomal metabolism of certain drugs and steroids in the rat is well established (Kato and Kamataki 1982). In general, sex differences become apparent at puberty and are associated with lower rates of metabolism in females compared to males toward the biotransformation of many substrates. The magnitude of observed sex differences is dependent upon the substrate and metabolic pathway studied, and microsomes from females catalyze certain reactions more efficiently than males (Gustafsson and Ingelman-Sundberg 1975). Age- and gender-related differences in metabolism are attributable, at least in part, to the cytochrome P450 composition of hepatic microsomes. The superfamily of cytochrome P450 isozymes (compiled by Nebert et al. 1991), which possess different but overlapping substrate selectivities, serve as the terminal oxidase and substrate binding site for a vast array of xenobiotics and endogenous compounds (Ryan and Levin 1990). One female-specific isozyme, cytochrome P450i (CYP2C12), and four male-specific isozymes, cytochromes P450 RLM2 (CYP2A2), P450g (CYP2C13), P450h (CYP2C11), and P4501 (CYP3A2), have been purified to homogeneity from rat liver microsomes (Cheng and Schenkman 1982; Kamataki et al. 1983b; Jansson et al. 1985; Ryan et al. 1984a; Halpert 1988; Cooper et al. 1993). Other cytochromes P450, such as cytochrome P450f (CYP2C7), are present in microsomes from females and males but are differentially expressed (Bandiera et al. 1986).

Monoclonal antibodies have been successfully isolated which are isozyme-specific for cytochrome P450p (3A1) or P4501 (3A2), two members of the steroid-inducible cytochrome P450 subfamily exhibiting 89% amino acid sequence homology, and these antibodies show less than 5% cross-reaction with 11 other cytochromes P450 (P450a-P450k). A library of 28 purified monoclonal antibodies was established and characterized as to epitope specificity. Appropriate antibodies were selected and utilized to investigate the regulation of expressed cytochrome P450p and P4501 proteins as a function of age, sex, and treatment of rats with various inducing agents. Cytochrome P450p is not detectable in hepatic microsomes from untreated immature or adult male and female rats. Following dexamethasone treatment, expression of cytochrome P450p is observed in all groups with the levels reaching 30-37% of total microsomal cytochrome P450. Administration of other inducers such as pregnenolone 16 alpha-carbonitrile also yield enhanced levels of cytochrome P450p. Measurable amounts of constitutive cytochrome P4501 were detected in hepatic microsomes from immature and adult males as well as immature females but not in adult females. Cytochrome P4501 expression is inducible by dexamethasone in immature rats of both sexes and adult males, although dexamethasone is more effective as an inducer of cytochrome P450p than cytochrome P4501. Hence, not only is cytochrome P4501 protein expressed in immature animals of both sexes, it is also inducible in both sexes. These studies show that constitutive expression and induction of steroid-inducible cytochrome P450s may vary as a function of age.

Monoclonal antibodies have been successfully isolated which are isozyme-specific for cytochrome P450p (3A1) or P450l (3A2), two members of the steroid-inducible cytochrome P450 subfamily exhibiting 89% amino acid sequence homology, and these antibodies show less than 5% cross-reaction with 11 other cytochromes P450 (P450a-P450k). A library of 28 purified monoclonal antibodies was established and characterized as to epitope specificity. Appropriate antibodies were selected and utilized to investigate the regulation of expressed cytochrome P450p and P450l proteins as a function of age, sex, and treatment of rats with various inducing agents. Cytochrome P450p is not detectable in hepatic microsomes from untreated immature or adult male and female rats. Following dexamethasone treatment, expression of cytochrome P450p is observed in all groups with the levels reaching 30-37% of total microsomal cytochrome P450. Administration of other inducers such as pregnenolone 16α-carbonitrile also yield enhanced levels of cytochrome P450p. Measurable amounts of constitutive cytochrome P450l were detected in hepatic microsomes from immature and adult males as well as immature females hut not in adult females. Cytochrome P450l expression is inducible by dexamethasone in immature rats of both sexes and adult males, although dexamethasone is more effective as an inducer of cytochrome P450p than cytochrome P450l. Hence, not only is cytochrome P450l protein expressed in immature animals of both sexes, it is also inducible in both sexes. These studies show that constitutive expression and induction of steroid-inducible cytochrome P450s may vary as a function of age.

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.

We have shown previously that 2,3,4,5-tetrachlorobiphenyl is ineffective as an inducer of rat liver microsomal cytochrome P-450. Addition of a single para-chloro substituent in the otherwise unsubstituted phenyl ring, to give 2,3,4,4',5-pentachlorobiphenyl, produces a potent cytochrome P-450 inducer with both phenobarbital- and 3-methylcholanthrene-type characteristics. In the present study, 2,3,4,5-tetrachlorobiphenyl was substituted in the para(4') position with 12 other functional groups. The 4'-X-C12H5Cl4 derivatives were tested as inducers of cytochromes P-450a--P-450e and epoxide hydrolase, by immunochemical analysis of liver microsomes prepared 4 days after a single treatment (500 mumol/kg) of 1-month-old male Long Evans rats. When the para' substituent was a halogen (F, Cl, Br or I), the derivative induced both cytochromes P-450b and P-450e, and cytochromes P-450c and P-450d, which are the major phenobarbital- and 3-methylcholanthrene-inducible isozymes, respectively. A similar type of induction was observed with a second group of derivatives substituted with CN, NO2 or CF3. However, a derivative containing CH3CO--(which is also a meta-directing, ring-activating substituent) failed to induce cytochromes P-450a-P-450e at the dosage and time tested. Members of a third group of derivatives, which contained an ortho/para-directing, ring-activating substituent) were either ineffective inducers (OH, CH3, CH3O--), or were inducers of cytochromes P-450c and P-450d (isopropyl or t-butyl). Hence, 4'-substitution with a bulky lipophilic substituent conferred 3-methylcholanthrene- but not phenobarbital-type characteristics on 2,3,4,5-tetrachlorobiphenyl. Some of the derivatives tested, namely those substituted with Cl, Br, I and CF3, were remarkably effective inducers of cytochrome P-450a, causing a 10-11-fold induction of this isozyme. Data on the induction of cytochrome P-450c were analyzed by multiparameter linear regression in an attempt to correlate the biological activity of the 4'-X-C12H5Cl4 derivatives with the physiochemical properties of the various substituents. From these results, and those reported recently, we propose that binding of the 4'-X-C12H5Cl4 derivatives to the rat cytosolic Ah receptor is favored by increasing the electronegativity, lipophilicity and hydrogen bonding characteristics of the 4' substituent, whereas enzyme induction (both in vivo and in cultured rat hepatoma cells) is also governed by a fourth characteristic, the STERIMOL factor, which gives a measure of the width of the substituent.(ABSTRACT TRUNCATED AT 400 WORDS)

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.

A simple, non-chromatographic purification procedure for monoclonal antibodies from mouse ascites fluid is described. This procedure, which is rapid, inexpensive, and has high capacity involves the precipitation of contaminating proteins with caprylic acid followed by precipitation of immunoglobulin using ammonium sulfate. This two-step procedure is shown to be effective for the purification of various immunoglobulins including IgG1, IgG2a and IgG2b. In the present report, more than 30 monoclonal antibodies directed against cytochrome P450 isozymes have been purified by this method and characterized.

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitrosodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%)NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti-P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti-P-450j to HLj. The addition of cytochrome b5 to reconstitution systems containing HLj resulted in a small increase in the Vmax for NDMA demethylation accompanied by a decrease in Km,app (1.3 to 0.3 mm) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cisacting.

The principal oxidative metabolites formed from benzo(c)phenanthrene (B(c)Ph) by the cytochromes P450 in liver microsomes from control and treated rats are the 3,4- and 5,6-arene oxides. A procedure is described which allows determination of the enantiomer composition and absolute configuration of these arene oxides based on HPLC separation of isomeric thiolate adducts formed with N-acetyl-L-cysteine in base. Incubation of [3H]-B(c)Ph with highly purified cytochrome P450c in a reconstituted monooxygenase system followed by trapping of the metabolically formed arene oxides as above indicated that the 3,4-oxide was predominantly the (+)-(3S,4R)-enantiomer (90%) and that the 5,6-oxide consisted mainly of the (+)-(5S,6R)-enantiomer (76%). The results are discussed in terms of their implications about the catalytic binding site of cytochrome P450c.

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17–18, 37–46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (>90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450f and P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (less than 1%) of cytochrome P450g and the other high levels (greater than or equal to 10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refractory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.

Numerous reports have illustrated the versatility of polychlorinated biphenyls (PCBs) and related halogenated aromatics as inducers of drug-metabolizing enzymes and the activity of individual compounds are remarkably dependent on structure. The most active PCB congeners, 3,4,4′,5-tetra-, 3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs resembled 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in their mode of induction of the hepatic drug-metabolizing enzymes. These compounds induced rat hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) and cytochromes P-450a, P-450c and P-450d. 3,4,4′,5-Tetrachlorobiphenyl, the least active coplanar PCB, also induced dimethylaminoantipyrine N-demethylase and cytochromes P-450b+e and resembled Aroclor 1254 as an inducer of the mixed-function oxidase system. Like Aroclor 1254, all the mono-ortho- and at least eight di-ortho-chloro analogs of the coplanar PCBs exhibited a "mixed-type" induction pattern and induced microsomal AHH, dimethylaminoantipyrine NM-demethylase and cytochromes P-450a–P-450e. Quantative structure–activity relationships (QSARs) within this series of PCBs were determined by comparing their AHH induction potencies (EC50) in rat hepatoma H-4-II-E cells and their binding affinities (ED50) for the 2,3,7,8-TCDD cytosolic receptor protein. The results showed that there was an excellent correlation between AHH induction potencies and receptor binding avidities of these compounds and the order of activity was coplanar PCBs (3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyls) > 3,4,4′,5-tetrachlorobiphenyl ~ mono-ortho coplanar PCBs > di-ortho coplanar PCBs. It was also apparent that the relative toxicities of this group of PCBs paralleled their biological potencies. The coplanar and mono-ortho coplanar PCBs also exhibit differential effects in the inbred C57BL/6J and DBA/2J mice. These compounds induce AHH and cause thymic atrophy in the former "responsive" mice whereas at comparable or higher doses none of these effects are observed in the nonresponsive DBD/2J mice. Since the responsiveness of these two mice strains is due to the presence of the Ah receptor protein in the C57BL/6J mice and its relatively low concentration in the DBA/2J mice, the results for the PCB cogeners support the proposed receptor-mediated mechanism of action. Although the precise structural requirements for ligand binding to the receptor have not been delineated, the halogenated aromatic hydrocarbons which exhibit the highest binding affinities for the receptor protein are approximate isostereomers of 2,3,7,8-TCDD. 2,3,4,4′,5-Pentachlorobiphenyl elicits effects which are qualitatively similar to that of TCDD and the presence of the lateral 4′-substituent is required for this activity. Thus the 4′-substituted 2,3,4,5-tetrachlorobiphenyls have been used as probes for determining the substituent characteristics which favor binding to the receptor protein. Multiple regression analysis of the competitive binding EC50 values for 13 substituents gave the following equation: log (1/EC50) = 1.53σ + 1.47π + 1.09 HB + 4.08 where σ is electronegativity, π is hydrophobicity, HB is hydrogen bonding and r is the correlation coefficient (r = 0.978). The utility of this equation in describing ligand:receptor interactions and correlations with toxicity are being studied with other halogenated hydrocarbons and PAHs.