Binding of 6-hydroxymethylbenzo[a]pyrene and 6-acetoxymethylbenzo[a]pyrene to DNA.

The carcinogenic hydrocarbons 6-hydroxymethylbenzo[a]pyrene (6-HOCH2-B[a]P) and 6-acetoxymethylbenzo[a]pyrene (6-AcOCH2-B[a]P) were examined for their ability to bind to rat and calf thymus DNA. The data indicate there are no appreciable differences in the amount of binding to the two types of DNA. Non-enzymatic binding of 6-HOCH2-B[a]P was low (5 mumol hydrocarbon/mol DNA P) but 6-AcOCH2-B[a]P was bound to a considerable extent (88.4--97.3 mumol hydrocarbon/mol DNA P). Non-enzymatic binding of 6-HOCH2-B[a]P was greatly increased in the presence of ATP. Binding of 6-HOCH2-B[a]P in the presence of liver microsomes from untreated rats or from rats pretreated with 3-methylcholanthrene (3-MC) never exceeded 5 mumol hydrocarbon/mol DNA P. Binding of 6-HOCH2-B[a]P in the presence of a PAPS generating system was less than non-enzymatic binding mediated by ATP and was dependent on the presence of ATP rather than ATP and sulfate. Binding was reduced by 50% when ADP was employed in the non-enzymatic reaction and was negligible in the presence of AMP or adenosine, indicating that a diphosphate group is necessary. Incubation of 6-HOCH2-B[a]P with DNA in the presence of ATP, CTP, GTP, or UTP showed that ATP was the most effective mediator of the binding reaction. These observations suggest that 6-HOCH2-B[a]P is converted to a phosphate ester which, like 6-AcOCH2-B[a]P, is much more reactive than 6-HOCH2-B[a]P itself.     

Asbestos catalyzes the formation of the 6-oxobenzo[a]pyrene radical from 6-hydroxybenzo[a]pyrene

Crocidolite asbestos catalyzes the oxidation of 6-hydroxybenzo[a]pyrene, a metabolite of benzo[a]pyrene, to the 6-oxobenzo[a]pyrene radical as determined by electron spin resonance spectroscopy. This may be a mechanism whereby inhaled asbestos enhances the incidence of lung cancer induced by cigarette smoke, which contains benzo[a]pyrene.     

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.     

Sulfate conjugation of benzo[alpha]pyrene metabolites and derivates

Sulfate conjugation of benzo[alpha]pyrene(BP) metabolites and derivatives was studied. The reaction sequence consisted of two steps; activation of sulfate ion to 3'-phosphoadenosine-5'-phosphosulfate and transfer of the activated sulfate to the BP-derivatives. Both reactions were carried out by enzymes located in the rat liver 105 000 g supernatant. The reactions required MgCl2. Phenol and quinone derivatives were generally good substrates for sulfate conjugation and different reactivities were observed with the dihydrodiol derivatives. Sulfate conjugates were more polar than their parent BP-derivatives and except for quinone conjugates were easily extracted with ethyl acetate. The role of sulfate conjugation in BP carcinogenesis is discussed.     

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.     

Conjugation of benzo[a]pyrene metabolites by freshwater green alga Selenastrum capricornutum

Benzo[a]pyrene (BaP) undergoes metabolic transformation in mammals via oxidative, hydrolytic, and conjugative processes; however, little is known concerning BaP conjugation in freshwater algae. It has been shown in this laboratory that BaP is metabolized by Selenastrum capricornutum via a dioxygenase pathway. This study describes the conjugation of BaP metabolites by a green alga, Selenastrum capricornutum. Cultures were exposed to 1160 micrograms/l [14C]BaP for 4 days at 23 degrees C under gold fluorescent lights on a diurnal cycle of 16 h light, 8 h dark. Of the total metabolites in the algal culture, 89% were present in media. BaP and non-conjugated metabolites were separated from conjugated metabolites by chromatography on neutral alumina columns using solvents of increasing polarity. Seventy-one percent of the BaP metabolites were conjugates of which 12.2%, 12.0% and 12.4% were sulfate ester and alpha- and beta-glucose conjugates, respectively. Conjugates that coeluted with sulfate esters were hydrolyzed with arylsulfatase, alpha- or beta-glucosidase; high performance liquid chromatography (HPLC) analysis indicated that the major product of each enzymatic hydrolysis was the 4,5-dihydrodiol (87.2, 69 and 53%, respectively). Eighty-six percent of the conjugates were acid labile following incubation for 2 h in 4 N HCl at 37 degrees C. To our knowledge this is the first demonstration of the metabolism of a polynuclear aromatic hydrocarbon by a freshwater green alga through a dioxygenase pathway and subsequent conjugation and excretion.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Human intestinal Caco-2 cells display active transport of benzo[a]pyrene metabolites

Epithelial cells of the gastrointestinal tract are challenged by exposure to many potentially toxic agents including the well-known food contaminant benzo[a]pyrene (B[a]P). They are equipped with a variety of Phase 1- and Phase 2-enzymes that are able to metabolize B[a]P. Furthermore, transmembranous ABC-transport proteins are expressed at the apical pole of these cells. The aim of this study was to investigate whether [14C]B[a]P or products of the metabolism are transported by intestinal cells back into the gut lumen. The intestinal Caco-2 cell line was used as a metabolism and transport model. Experiments with Caco-2 monolayers in the Transwell-system revealed that radiolabeled substance is transported towards the apical (luminal) region. This transport was characterized as active and increased after induction of cytochromes P450 1A1 and 1B1 by beta-naphthoflavone. On the other hand, transport was decreased with the concomitant inhibition of Phase 1-metabolism. TLC-analysis revealed that the primary metabolites of B[a]P found in the supernatant were very polar; other metabolites of less polarity could only be detected in trace amounts. These results indicate that B[a]P is metabolized by Caco-2 cells to highly polar metabolites resulting from biphasic metabolism and that these polar metabolites are subject to an apically directed transport. Chemical inhibition studies showed that P-glycoprotein and MRP1 or 2 were not involved in this polarized B[a]P-metabolite secretion.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

Effects of pretreatment with benzo[a]pyrene on the stereochemical selectivity of metabolic activation of benzo[a]pyrene to DNA-binding metabolites in hamster embryo cell cultures

The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.     

A fluorometric-HPLC assay for quantitating the binding of benzo[a]pyrene metabolites to DNA

A nonradiometric method is presented for quantitating low levels of benzo[a]pyrene (BP) derivatives that are covalently bound to the DNA of BP-treated mice. This method consists of hydrolyzing the DNA with acid to liberate the BP-adducts in the form of the isomeric tetrols of BP. These tetrols have fluorescence quantum yields of ～0.7 in deoxygenated solution at 298 K. Hence they are easily quantitated, following HPLC separation, by means of fluorescence detection. The sensitivity of the method is such that one bound BP residue per 10⁷ bases can be detected in 100 μg of DNA.     

Co-mutagenic activity of arsenic and benzo[a]pyrene in mouse skin

Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenic-induced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.     

Formation of DNA-binding products from isolated benzo[a]pyrene metabolites in rat liver nuclei

Liver nuclei from 3-methylcholanthrene-treated rats in the presence of NADPH metabolized 3- and 9-hydroxybenzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to products that bound to DNA. Maximal binding was obtained with the dihydrodiol which was approximately 3-fold that with 9-hydroxybenzo[a]pyrene, and 60-fold that with 3-hydroxybenzo[a]pyrene, as substrates. Both 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene and 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene were also extensively metabolized by the nuclear fraction but did not give rise to DNA-binding products.

The available evidence suggests that the DNA binding species derived from 9-hydroxy-benzo[a]pyrene is 9-hydroxy-benzo[a]pyrene-4,5-oxide and from 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, as previously observed in different systems, 7,8-dihydro-7,8-dihydroxy-benzo[a]pyrene-9,10-oxide.     

Hydroxymethylation of the benzene ring. Microsomal hydroxymethylation of benzo(alpha)pyrene to 6-hydroxymethylbenzo(alpha)pyrene

The formation of 6-hydroxymethylbenzo(α)pyrene from benzo(α)pyrene has been shown to be catalyzed by sonicates of rat liver microsomes. The biosynthetic pathway for the formation of the aryl hydroxymethyl groups appears to be a direct hydroxymethylation of the benzene ring and not involve 6-methylbenzo(α)pyrene as an intermediate, because the formation of 6-hydroxymethylbenzo(α)pyrene is not a cytochrome P-450-mediated reaction, whereas a model aryl side chain methyl group hydroxylation has been shown to be inhibited by cytochrome P-450 inhibitors.     

Nitroreduction of 6-nitrobenzo[a]pyrene: a potential activation pathway in humans

6-Nitrobenzo[a]pyrene, an environmental pollutant, was metabolized by human intestinal microflora to 6-nitrosobenzo[a]pyrene and 6-aminobenzo[a]pyrene. The two-electron reduction product 6-nitrosobenzo[a]pyrene exhibited strong direct-acting mutagenicity in the Salmonella typhimurium assay. These results imply that 6-nitrobenzo[a]pyrene can be hazardous to human health via a nitroreduction activation pathway and opens the possibility that other nitro-polycyclic aromatic hydrocarbons that are not direct-acting mutagens may be activated in vivo by a similar mechanism.