Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity

C57BL/6N (Ahb/Ahb) mice have a high-affinity Ah receptor in tissues, whereas AKR/J and DBA/2N (Ahd/Ahd) mice have a poor-affinity Ah receptor. The cytochrome P1-450 induction response (enhanced benzo[a]pyrene metabolism) occurs much more readily in Ahb/Ahb and Ahb/Ahd than in Ahd/Ahd mice, at any given dose of the inducer benzo[a]pyrene. Embryos from the AKR/J X (C57BL/6N)(AKR/J)F1 and the reciprocal backcross were studied during benzo[a]pyrene feeding of the pregnant females. Oral benzo[a]pyrene (120 mg/kg/day) given to pregnant Ahd/Ahd mice between gestational day 2 and 10 produces more intrauterine toxicity and malformations in Ahd/Ahd than Ahb/Ahd embryos. This striking allelic difference is not seen in pregnant Ahb/Ahd mice receiving oral benzo[a]pyrene. Pharmacokinetics studies with [3H]benzo[a]pyrene in the diet and high-performance liquid chromatographic analysis of benzo[a]pyrene metabolism in vitro by the maternal intestine, liver, and ovary and the embryos of control and oral benzo[a]pyrene-treated pregnant females are consistent with "first-pass elimination" kinetics and differences in benzo[a]pyrene metabolism by the embryos and/or placentas versus maternal tissues. In the pregnant Ahd/Ahd mouse receiving oral benzo[a]pyrene, little induction of benzo[a]pyrene metabolism occurs in her intestine and liver; this leads to much larger amounts of benzo[a]pyrene reaching her embryos, and genetic differences in toxicity and teratogenesis are manifest. In the pregnant Ahb/Ahd mouse receiving oral benzo[a]pyrene, benzo[a]pyrene metabolism is greatly enhanced in her intestine and liver; this leads to less benzo[a]pyrene reaching her embryos, much less intrauterine toxicity and malformations, and no genetic differences are manifest. More toxic metabolites (especially benzo[a]pyrene 1,6- and 3,6-quinones) are shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos. In additional studies, no prenatal or neonatal "imprinting" effect in C57BL/6N mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 on benzo[a]pyrene metabolism later in life was detectable. These genetic differences in intrauterine toxicity and teratogenicity induced by oral benzo[a]pyrene are just opposite those induced by intraperitoneal benzo[a]pyrene [Shum et al., '79; Hoshino et al., '81). The data in the present report emphasize the importance of the route of administration when the teratogen induces its own metabolism.     

Inhibition of binding of benzo(a)pyrene to DNA by 7,8-benzoflavone in organ cultures of human bronchus

Levels of binding of exogeneously added benzo(a)pyrene to DNA in organ culture were examined in nine specimens of normal human bronchus obtained by bronchoscopy of tumor patients. The specimens were divided into two portions and incubated with [3H]benzo(a)pyrene in the absence or presence of 2 microM 7,8-benzoflavone for 24 h. 7,8-benzoflavone inhibited [3H]benzo(a)pyrene-DNA binding from 24 to 60%. Generally, the levels of binding of [3H]benzo(a)pyrene to DNA in the presence of 7,8-benzoflavone were relatively low and closely bracketed the mean value for the nine specimens. This appears to indicate that there are at least two components to [3H]benzo(a)pyrene-DNA binding catalyzed by the human bronchus. One component is quite variable in activity and is sensitive to inhibition by 7,8-benzoflavone, and may be an environmentally induced activity. The second component is lower in activity, and may be a constitutive portion of the mixed-function oxidase.     

Isolation and characterization of rat liver cytosolic aldehyde dehydrogenases induced by phenanthrene or benzo[a]pyrene

The cytosolic aldehyde dehydrogenase was isolated from the liver of Wistar rats treated with phenanthrene (non-carcinogenic) or benzo[a]pyrene (carcinogenic polycyclic aromatic hydrocarbon). The benzo[a]pyrene-induced enzyme has higher Km values for small aliphatic aldehydes and a lower molecular weight than the phenanthrene-induced enzyme. It is more resistant to changes of pH and to inhibition by disulfiram, but more sensitive to heat denaturation than the phenanthrene-induced enzyme. The phenanthrene-induced aldehyde dehydrogenase is very similar to the normal uninduced aldehyde dehydrogenase, whereas the benzo[a]pyrene-induced aldehyde dehydrogenase has common properties with the TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced enzyme and the hepatoma-specific enzyme.     

Metabolism of benzo[a]pyrene: Effect of 3-methylcholanthrene pretreatment on metabolism by microsomes from lungs of genetically “responsive” and ”nonresponsive” mice

The metabolism of [¹⁴C]benzo[a]pyrene by microsomes from the lungs of normal and 3-methylcholanthrene-treated DBA/2J, C57BL/6J, and A/HeJ mouse strains was quantitatively analyzed by high-pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with lung microsomes than with liver microsomes in all three strains. The ratio of epoxide hydrase to monooxygenase activity in mouse lung was shown to be considerably higher than in mouse liver. Benzo[a]pyrene metabolism by control lung microsomes showed some strain differences. C57BL/6J and A/HeJ mice formed twice as much dihydrodiols as a percentage of total metabolism compared to DBA/2J mice. DBA/2J mice produced somewhat less phenol 2 fraction and considerably more quinone 1 and 2 fractions than the other two mouse strains as a percentage of total metabolism. Treatment of C57BL/6J and DBA/2J mice with 3-methylcholanthrene resulted in a 20-fold increase in the metabolism of benzo[a]pyrene, while A/HeJ mice were induced more than 50-fold. The profiles of metabolites from the 3-methylcholanthrene-induced animals were nearly identical in all three mouse strains.     

Bacterial mutagenicity of two cyclopentafused isomers of benzpyrene

Two novel cyclopentafused polycyclic aromatic hydrocarbons, naphtho(1,2,3-mno)acephenanthrylene (cyclopenta benzo[e]pyrene) and naphtho(2,1,8-hij)acephenanthrylene (cyclopenta(ij)benzo[a]pyrene) were evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. Both compounds required S9 metabolic activation, and showed optimal activity at low S9 concentrations (below 0.6 mg/plate). Both compounds induced frameshift and base-pair substitution mutations, being active in strains TA98, TA100, TA1537, TA1538 and TA104, but not in strain TA1535. Cyclopenta(ij)benzo[a]pyrene was more active than cyclopentabenzo[e]pyrene, and both were more potent than their parent ring systems, benzo[a]pyrene and benzo[e]pyrene, respectively. Cyclopenta(ij)benzo[a]pyrene was more active in strain TA104 than in TA100 or TA98 (250-470, 340 and 80-100 rev/nmole) as was benzo[a]pyrene (120, 70 and 40 rev/nmole respectively); cyclopentabenzo[e]pyrene was more active in TA100 than TA104 or TA98 (70 versus 50 and 40 rev/nmole), and benzo[e]pyrene showed a similar pattern (4, 3.5 and 0.6 rev/nmole). The relative potencies of the four compounds are in accord with predictions based on perturbational molecular orbital calculations. The peak of activity at low S9 concentrations is consistent with epoxidation at the cyclopentafused ring being the major route of metabolic activation for both these cyclopentafused compounds.     

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 24h exposure assay, the highest IEFs were found for benzo[k]fluoranthene, dibenzo[a,h]anthracene and dibenzo[a,k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[a,j]anthracene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene, and naphtho[2,3-a]pyrene. The 6h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[a,l]pyrene, cyclopenta[cd]pyrene, and benzo[a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[k]fluoranthene and benzo[j]fluoranthene, followed by indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[a]pyrene, dibenzo[a,j]anthracene, chrysene, and benzo[b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[a]pyrene, dibenzo[a,e]pyrene, and naphtho[2,3-a]pyrene and to a lesser extent also for benzo[a]anthracene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene and dibenzo[a,i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.     

The homogeneity of rat liver microsomal cytochrome P-448 activity and its role in the activation of benzo[a]pyrene to mutagens

The O-deethylation of ethoxyresorufin and the metabolic activation of benzo[a]pyrene to mutagens were determined in hepatic microsomal preparations from control and induced animals. An excellent direct correlation (r = 0.95) has been observed between ethoxyresorufin O-deethylase and the metabolic activation of benzo[a]pyrene to mutagens when the fraction of cytochromes P-450 present as cytochrome P-448 was altered by the administration of phenobarbitone and 3-methylcholanthrene alone or in combination with 9-hydroxyellipticine. The correlation between these activities was maintained following treatment of animals with Arochlor 1254, benzo[a]pyrene, benzo[e]pyrene, 7,12-dimethylbenzo[a]anthracene,2-anthramine and 2-naphthylamine.     

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.     

Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyrene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 microM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 microM). At pH 7.0, the specificity constant ranged from (1.3 +/- 0.1) x 10(6) M(-1) s(-1) with benz[a]anthracene 1,2-dihydrodiol to (20.0 +/- 0.8) x 10(6) M(-1) s(-1) with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.     

Potency of various polycyclic aromatic hydrocarbons as inducers of CYP1A1 in rat hepatocyte cultures

A number of highly toxic environmental pollutants including certain polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and 'dioxin-like' polychlorinated biphenyls (PCB) are among the most potent agonists of the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A1 (CYP1A1) in mammalian cell culture is widely used as a functional parameter for AHR activation providing an estimate for 'dioxin-like' inducing equivalents in extracts from environmental samples. Since a number of polycyclic aromatic hydrocarbons (PAHs) also act as AHR-agonists, the CYP1A1-inducing potencies, measured as induction of 7-ethoxyresorufin O-deethylase (EROD) activity in rat hepatocyte cultures were analyzed for 16 PAHs frequently present in environmental samples. Among these, seven PAHs including benzo[a]pyrene were relatively potent inducers allowing the determination of Induction Equivalency Factors (IEF). For three PAHs including benzo[k]fluoranthene which acted as weak inducers, IEFs were estimated, while six PAHs including acenaphthylene were classified as inactive. Based on different efficacies the concentration-response characteristics of CYP1A1 induction were analyzed in more detail for benzo[a]pyrene, benzo[k]fluoranthene, and acenaphthylene. Benzo[k]fluoranthene was markedly less effective than benzo[a]pyrene as inducer of EROD activity but even more effective than benzo[a]pyrene as inducer of CYP1A1 protein and mRNA. Acenaphthylene was highly more effective on the level of mRNA than on the levels of protein or EROD activity. Further analysis revealed that the low efficacy of acenaphthylene as inducer of CYP1A1 protein and EROD activity is due to its marked cytotoxicity while no clear-cut explanation was found for the differences in efficacy between benzo[k]fluoranthene and benzo[a]pyrene. The EROD-inducing potency of a mixture of 16 PAH was about 2-fold higher than that calculated on the basis of IEFs of the individual constituents of the mixture.     

Rhodanobacter sp. strain BPC1 in a benzo[a]pyrene-mineralizing bacterial consortium

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.     

Differential response of human and rat epoxide hydrolase to polycyclic aromatic hydrocarbon exposure: studies using precision-cut tissue slices

The potential of polycyclic aromatic hydrocarbons (PAHs) to modulate microsomal epoxide hydrolase activity, determined using benzo[a]pyrene 5-oxide as substrate, in human liver, was evaluated and compared to rat liver. Precision-cut liver slices prepared from fresh human liver were incubated with six structurally diverse PAHs, at a range of concentrations, for 24 h. Of the six PAHs studied, benzo[a]pyrene, dibenzo[a,h]anthracene and fluoranthene gave rise to a statistically significant increase in epoxide hydrolase activity, which was accompanied by a concomitant increase in epoxide hydrolase protein levels determined by immunoblotting. The other PAHs studied, namely dibenzo[a,l]pyrene, benzo[b]fluoranthene and 1-methylphenanthrene, influenced neither activity nor enzyme protein levels. When rat slices were incubated under identical conditions, only benzo[a]pyrene and dibenzo[a,h]anthracene elevated epoxide hydrolase activity, which was, once again accompanied by a rise in protein levels. At the mRNA level, however, all six PAHs caused an increase, albeit to different extent. In rat, epoxide hydroxylase activity in lung slices was much lower than in liver slices. In lung slices, epoxide hydrolase activity was elevated following exposure to benzo[a]pyrene and dibenzo[a,l]pyrene and, to a lesser extent, 1-methylphenanthrene; similar observations were made at the protein level. At both activity and protein levels extent of induction was far more pronounced in the lung compared with the liver. It is concluded that epoxide hydrolase activity is an inducible enzyme by PAHs, in both human and rat liver, but induction potential by individual PAHs varies enormously, depending on the nature of the compound involved. Marked tissue differences in the nature of PAHs stimulating activity in rat lung and liver were noted. Although in the rat basal lung epoxide hydrolase activity is much lower than liver, it is more markedly inducible by PAHs.     

A simple radioisotope assay for microsomal aryl hydroxylase

A radioassay for liver microsomal aryl hydroxlase activity has been devised which depends on the liberation of tritiated water from generally tritiated benzo[α]pyrene during hydroxylation. The quantity of tritiated water has been shown to be proportional to the amount of 3-hydroxybenzo-[α]pyrene formed. Among the advantages of the radioassay are its speed, simplicity, and the fact that it essentially provides a cumulative measure of the hydroxylation of the benzo[α]pyrene ring. Investigation of a number of variables has made it possible to assay and obtain proportional results with as little as 3 μg of rat liver microsomes. Nucleic acids, but not their component mononucleotides, have been found capable of protecting the enzyme from product inhibition, presumably by interaction with benzo[α]-pyrene oxide, the primary product of benzo[α]pyrene hydroxylation.     

DNA adducts formation and induction of apoptosis in rat liver epithelial 'stem-like' cells exposed to carcinogenic polycyclic aromatic hydrocarbons

The bipotent liver progenitor cells, so called oval cells, may participate at the early stages of hepatocarcinogenesis induced by chemical carcinogens. Unlike in mature parenchymal cells, little is known about formation of DNA adducts and other genotoxic events in oval cells. In the present study, we employed spontaneously immortalized rat liver WB-F344 cell line, which is an established in vitro model of oval cells, in order to study genotoxic effects of selected carcinogenic polycyclic aromatic hydrocarbons (PAHs). With exception of dibenzo[a,l]pyrene, and partly also benzo[g]chrysene and benz[a]anthracene, all other PAHs under the study induced high levels of CYP1A1 and CYP1B1 mRNA. In contrast, we observed distinct genotoxic and cytotoxic potencies of PAHs. Dibenzo[a,l]pyrene, and to a lesser extent also benzo[a]pyrene, benzo[g]chrysene and dibenzo[a,e]pyrene, formed high levels of DNA adducts. This was accompanied with accumulation of Ser-15 phosphorylated form of p53 protein and induction of apoptosis. Contrary to that, benz[a]anthracene, chrysene, benzo[b]fluoranthene and dibenzo[a,h]anthracene induced only low amounts of DNA adducts formation and minimal apoptosis, without exerting significant effects on p53 phosphorylation. Finally, we studied effects of 2,4,3',5'-tetramethoxystilbene and fluoranthene, inhibitors of CYP1B1 activity, which plays a central role in metabolic activation of dibenzo[a,l]pyrene. In a dose-dependent manner, both compounds inhibited apoptosis induced by dibenzo[a,l]pyrene, suggesting that it interferes with the metabolic activation of the latter one. The present data show that in model cell line sharing phenotypic properties with oval cells, PAHs can be efficiently metabolized to form ultimate genotoxic metabolites. Liver progenitor cells could be thus susceptible to this type of genotoxic insult, which makes WB-F344 cell line a useful tool for studies of genotoxic effects of organic contaminants in liver cells. Our results also suggest that, unlike in mature hepatocytes, CYP1B1 might be a primary enzyme responsible for formation of DNA adducts in liver progenitor cells.     

The strong hepatocarcinogenicity of the electrophilic and mutagenic metabolite 6-sulfooxymethylbenzo[a]pyrene and its formation of benzylic DNA adducts in the livers of infant male B6C3F1 mice

6-Hydroxymethylbenzo[a]pyrene was activated to an electrophilic and mutagenic sulfuric acid ester metabolite by rat and mouse liver sulfotransferase activity. The intrinsic mutagenicity of this reactive ester, 6-sulfooxymethylbenzo[a]pyrene, was inhibited by glutathione and glutathione S-transferase. A single i.p. dose of 2.5 nmol/g body wt of 6-sulfooxymethylbenzo[a]pyrene in infant male B6C3F1 mice induced liver tumors in 35 of 36 mice at 10 months with an average multiplicity of 4.4. A comparable dose of the parent hydrocarbon, 6-hydroxymethylbenzo[a]pyrene, was only a tenth as active. The electrophilic sulfuric acid ester produced high levels of benzylic DNA adducts in the livers of these mice that accounted for about 80% of the total DNA adducts. These results strongly suggest that this sulfuric acid ester is an important ultimate electrophilic and carcinogenic metabolite in carcinogenesis by 6-hydroxymethylbenzo[a]pyrene and possibly even by 6-methylbenzo[a]pyrene and benzo[a]pyrene in mouse liver.     

Effects of harman and norharman on the mutagenicity and binding to DNA of benzo[a]pyrene metabolites in vitro and on aryl hydrocarbon hydroxylase induction in cell culture

Harman and norharman, two β-carboline derivatives known to exist in certain foods and to be formed during pyrolysis of tobacco and meat, were tested for mutagenic activity in the presence of benzo[a]pyrene, mouse liver enzymes, and Salmonella typhimurium TA98 in vitro. Both harman and norharman inhibit benzo[a]pyrene mutagenicity, benzo[a]pyrene metabolism (as measured by aryl hydrocarbon hydroxylase activity), and the binding of all benzo[a]pyrene metabolites to DNA in vitro. Moreover, harman and norharman are quite toxic to cultures of hepatoma-derived H-4-II-E and Hepa-1 established cell lines and therefore were found to be very weak inducers of aryl hydrocarbon hydroxylase activity.     

Distribution and macromolecular binding of benzo[a]pyrene and two polychlorinated biphenyl congeners in female mice

PCBs are complete rodent carcinogens and their potent tumor promoting activity has been reported, but their tumor-initiating activity remains controversial. Macromolecular binding of PCB metabolites has been demonstrated in vitro, but this issue remains unclear in vivo. The purpose of this study was to determine the binding affinity of 4-chlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl to proteins and DNA in vivo. C57/BL6 female mice were treated intraperitoneally with hepatic enzyme inducers (phenobarbital and beta-naphthoflavone) and then with 14C-labelled polychlorinated biphenyls or benzo[a]pyrene. The short-term distribution of labeled compounds into liver, lungs and kidneys and into different sub-cellular fractions of these tissues was assessed and the DNA and proteins from the 700 x g pellet were further purified to assess covalent binding. All compounds were distributed in low amounts into the liver, kidneys and lungs, with the greatest accumulation in the liver, and the lowest in lungs. In all tissues, test compounds were mostly found in cytosols and organellar pellets (10,000 x g), and lower amounts were present in nuclear pellets (700 x g) and microsomes. In lungs and kidneys, only benzo[a]pyrene showed significant covalent binding to proteins. In the liver, protein binding indices were significant for all compounds (P<0.05), but no significant binding of the test compounds to DNA could be demonstrated with this approach. Our results suggest that at the 24 h time point, all compounds were activated to electrophilic intermediates prone to macromolecular binding. Hepatic proteins apparently act as a sink for PCB-derived electrophiles, thus preventing detectable levels of covalent binding to hepatic DNA or to proteins in less metabolically active tissues.     

New approach to synthesis of peptide immunomimetic of benzo[a]pyrene

A novel approach to discovery of the peptide with an internal immunological image of carcinogen is suggested in this work. The hybridomas producing monoclonal antibodies (mAb) B2 against benzo[a]pyrene and benz[a]antracene were prepared. Polyclonal Ab against benzo[a]pyrene (Bp) and benz[a]antracene (Ba), antracene, chrysene, and pyrene were also prepared. We identified the related peptide-presenting phage specificity binding to mAT B2 and polyclonal Ab against Bp from 12 large random peptide libraries using phage display method. ICR mice were immunized with specific binding of positive phage clone for studying its immunogenicity. The Bp antibodies were found in the blood serum. All positive phage clones carried the sequence. Thus, the unique peptide with an internal immunological image of Bp may be the new candidate for anticarcinogenic vaccine.     

Regio- and stereospecificity of homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons

The homogeneous 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol dehydrogenase EC 1.3.1.20), Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). Examination of the substrate specificity of the purified dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons indicates that the enzyme will catalyze the NAD(P)-dependent oxidation of trans-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, 5-methylchrysene, and benzo[a]pyrene under physiological conditions. Comparison of the utilization ratios Vmax/Km indicates that benzenedihydrodiol and the trans-1,2- and trans-7,8-dihydrodiols of 5-methylchrysene were most efficiently oxidized by the purified dehydrogenase, followed by the trans-7,8-dihydrodiol of benzo[a]pyrene and the trans-1,2-dihydrodiols of phenanthrene, chrysene, and naphthalene. The purified enzyme appears to display rigid regio-selectivity, since it will readily oxidize non-K-region trans-dihydrodiols but will not oxidize the K-region trans-dihydrodiols of phenanthrene and benzo[a]pyrene. The stereochemical course of enzymatic dehydrogenation was investigated by circular dichroism spectrometry. For the trans-1,2-dihydrodiols of benzene, naphthalene, phenanthrene, chrysene, and 5-methylchrysene, the dehydrogenase preferentially oxidized the (+)-[S,S]-isomer. Apparent inversion of this stereochemical preference occurred with the trans-7,8-dihydrodiol of 5-methylchrysene, as the (-)-enantiomer was preferentially oxidized. No change in the sign of the Cotton Effect was observed following oxidation of the racemic trans-7,8-dihydrodiol of benzo[a]pyrene, suggesting that both stereoisomers of this compound were substrates. Large-scale incubation of the [3H]-(+/-)-trans-7,8-dihydrodiol of benzo[a]pyrene with the purified dehydrogenase resulted in greater than 90% utilization of this potent proximate carcinogen, suggesting that the enzyme utilizes both the (-)-[R,R] and the (+)-[S,S]-stereoisomers, which confirms the circular dichroism result. These data show that dihydrodiol dehydrogenase displays the appropriate regio- and stereospecificity to catalyze the oxidation of both the major and minor non-K-region trans-dihydrodiols that arise from the microsomal metabolism of benzo[a]pyrene in vivo.