Effects of sulfite on the uptake and binding of benzo[a]pyrene diol epoxide in cultured murine respiratory epithelial cells.

Sulfur dioxide (SO2) may act as a cocarcinogen with benzo[a]pyrene (BaP) in the respiratory tract. We have modeled this effect by examining the interactions of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with sulfite, the physiological form of SO2, in a murine respiratory epithelial cell line (C10). We exposed C10 cells to [3H]-anti-BPDE and determined the effects of 1 and 10 mM sulfite on the uptake and subcellular localization of labeled products. Autoradiographic analysis showed that sulfite doubled the nuclear localization of anti-BPDE-derived materials after a 4-hr incubation period. The net nuclear localization of anti-BPDE-derived materials was not affected by sulfite during the first 60 min, but nuclear localization continued to increase in the sulfite-containing incubations throughout the 4-hr incubation period. Little increase in nuclear localization of anti-BPDE-derived material was noted in the incubations without sulfite after 60 min. Subcellular fractionation was performed to determine the amount of label associated with cytosolic and nuclear fractions and to determine covalent binding to protein and DNA. Sulfite produced a modest increase in the amount of [3H]-anti-BPDE-derived products bound to protein; however, binding to nuclear DNA increased by more than 200% with 10 mM sulfite. Analysis of the supernatants from the cytosolic and nuclear fractions of cells exposed to anti-BPDE and sulfite demonstrated the presence of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10c-su lfonate (BPT-10-sulfonate). [3H]-BPT-10-sulfonate was unable to enter C10 cells, suggesting that it is formed intracellularly.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin H synthase-dependent genotoxicity of 2,4-diaminotoluene.

2,4-Diaminotoluene (2,4-DAT), a high volume synthetic compound, is moderately carcinogenic to rodents. We report here that 2,4-DAT is a substrate for the peroxidase activity of prostaglandin H synthase (PHS). In contrast to many aromatic amines which are activated as mutagens by PHS, we find that 2,4-DAT is not mutagenic to six S. typhimurium strains with this activation system. The strains tested include YG1006, YG1024, and YG1029, which are far more sensitive to the mutagenicity of aromatic amines and nitroarenes than are the standard tester strains. Although not mutagenic itself, 2,4-DAT does enhance the mutagenicity of 2-aminofluorene (2-AF) in the PHS-catalyzed system in strains TA98, YG1006, and YG1024, with maximal enhancement of 140%, 1831%, and 1216%, respectively. Half-maximal enhancement of 2-AF mutagenicity is observed at 15-20 microM 2,4-DAT for strains YG1006 and YG1024, and about 80 microM for TA98. Studies with compounds structurally related to 2,4-DAT revealed enhancement of 2-AF mutagenicity with 2,5-DAT and o-phenylenediamine (o-PD) but not for other DAT isomers, toluidines, and phenylenediamines. Maximal enhancement of 2-AF mutagenicity observed in TA98 with PHS-catalyzed activation was 110% for o-PD and 60% for 2,5-DAT. This comutagenic effect of 2,4-DAT appears quite specific for 2-AF, as it fails to enhance either the PHS-dependent mutagenicity of the aromatic amines benzidine and 2-naphtylamine, or the direct mutagenicity of N-acetoxy-acetylaminofluorene,2-nitrofluorene,4- nitroquinoline-N-oxide and 1,1,1-trichloropropene-2,3-oxide. Enhancement of 2-AF mutagenicity by 2,4-DAT is also observed with cytochrome P-450-dependent activation, however the half-maximal 2,4-DAT concentration was 400 microM, and the maximal enhancement was only 50%. The ability of 2,4-DAT, under conditions where it is not itself mutagenic, to enhance the genotoxicity of the potent carcinogen 2-AF comprises an intriguing toxicological interaction, and underscores the inherent difficulties in assessing the genotoxic risks posed by mixtures of compounds.

Co-oxidation of xenobiotics: lipid peroxyl derivatives as mediators of metabolism.

Systems which carry out peroxyl-dependent oxidations can serve as activation systems for carcinogenic compounds. Some function via classical peroxidase reactions in which an enzyme-derived oxidant performs the electron abstraction from or oxygen donation to the oxidizable substrate. This mechanism applies to the peroxidative activation of aromatic amines and of the phenolic compound diethylstilbestrol. These classical peroxidase reactions may be initiated by hydrogen peroxide or by organic peroxides, including lipid hydroperoxides. A different mechanism is involved in the oxygenation of polycyclic aromatic hydrocarbons and of aflatoxin B1. In these cases the oxidant is a peroxyl radical, and the reaction occurs by the direct, non-enzymatic interaction of the peroxyl radical and the oxidizable substrate. Most peroxyl radicals in biological systems are lipid-derived. The key reaction which distinguishes the peroxyl radical-dependent oxidations from the classical peroxidase reactions is the ability of the former to epoxidize activated carbon-carbon double bonds. The epoxidation of benzo[a]pyrene derivatives has been studied extensively in subcellular and whole cell and tissue systems, and is discussed as a model for this class of reaction. Determining the generality of this activation path and its role in vivo present the major questions to be answered in regard to the importance of these reactions in chemical carcinogenesis.

Metabolic and genotoxic interactions of 2-aminofluorene and 2,4-diaminotoluene.

We have reported previously that the rodent carcinogen 2,4-diaminotoluene (2,4-DAT) is not activated as a mutagen to the standard Ames S. typhimurium tester strains when oxidized by prostaglandin H synthase (PHS). 2,4-DAT does, however, enhance the bacterial mutagenicity of the potent mutagen 2-aminofluorene (2-AF) when both compounds are incubated with the PHS activating system. Enhancement of activation of 2-AF would provide a plausible mechanism for the observed co-mutagenicity of 2,4-DAT. Co-incubation with 100 microM 2,4-DAT, however, inhibited the total metabolism of 25 microM 2-AF by 60% in both the PHS/H2O2 system and PHS/arachidonic acid system. The inhibition included a 75% decrease in the formation of water-soluble and protein-bound metabolites and about a 35% decrease in production of the peroxidative metabolites 2-nitrofluorene (NF) and 2-aminodifluorenylamine (ADFA). Azofluorene (AzF) production was the most sensitive to the effects of 2,4-DAT, exhibiting an 80% decrease in both PHS-catalyzed systems. No new 2-AF derived products were observed in the presence of 2,4-DAT. This pronounced inhibition of 2-AF metabolism by 2,4-DAT also was observed in incubations of the aromatic amines with PHS in the presence of S. typhimurium strain TA98. Bacterial N-acetylation of 2-AF did not appear to be an important reaction in any of these incubations. 2,4-DAT not only inhibited 2-AF metabolism by PHS, but also decreased the level of 2-AF covalent binding to the bacterial DNA by as much as 81%. This stands in sharp contrast to the enhancement of the mutagenicity of 2-AF elicited by 2,4-DAT in these same incubations. This clear dissociation between the extent of peroxidative activation, and resultant covalent modification of bacterial DNA, by 2-AF and the subsequent mutagenic response indicates that a metabolic interaction is not involved in the co-mutagenicity of 2,4-DAT.

An inexpensive system to monitor air flow in isolation units.

Isolation units are used extensively for conducting infectious disease research in poultry. By necessity, these units are airtight and receive air only through electrically powered ventilation systems. Therefore, interruptions in electrical service to these units present a serious hazard to the animals they contain. A system was designed to monitor the air flow through isolation units and to alert animal caretakers in the event of any interruption in air flow. The "intelligence" of the system relies on an electronic monitor connected to a telephone line that places alerting telephone calls when it detects loss of air flow to the units. The system is constructed from easily acquired and relatively inexpensive parts and components.

Efficacy of a novel copper-based footbath preparation for the treatment of ovine footrot during the spread period.

OBJECTIVE: To determine the efficacy of a novel copper based footbath preparation (CHF-1020) for treatment of ovine footrot during the spread period. DESIGN: A series of field trials with treated and control groups run together. ANIMALS: Mobs of at least 125 sheep on each of six properties in southern New South Wales with equal numbers of controls. PROCEDURE: Sheep of group A were treated after minimal paring by making them stand in CHF-1020 for 15 minutes. Treatment was undertaken at intervals throughout the period of the trials (14 September to 17 December 1993). Group A sheep were run on the same pasture as those from group B (untreated sheep). RESULTS: The percentage of sheep exhibiting clinical signs of ovine footrot at the start of the trial ranged from 35 to 88% at score 3 or higher, using a 0 to 5 footscoring system. During the trial, the percentage of infected sheep (greater or equal to score 2) in group B increased and ranged from 40 to 90%. The level of infected sheep in group A on each property was reduced progressively to 1 to 16%. Cure rates of 45 to 94% were achieved, with the lowest rate being on a property with a metal footbath. The next lowest cure rate was 73%. Results indicated that treatment should be undertaken at 2-weekly intervals while spread continues. Treated sheep can be returned to contaminated pastures. CONCLUSION: CHF-1020 is effective during the spread period and can be used for the progressive eradication of ovine footrot.

Sulfite-dependent mutagenicity of benzo[a]pyrene derivatives.

Benzo[a]pyrene (BP) and sulfur dioxide (SO2) are ubiquitous air pollutants and are also components of tobacco smoke. Although SO2 itself is not carcinogenic, concurrent administration with BP results in enhancement of respiratory tract tumorigenesis. In biological systems, SO2 exists as its hydrated form, sulfite (SO3(2-) ). Sulfite readily undergoes autoxidation, generating potent oxidant species. When 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is included in sulfite autoxidation mixtures it is converted to more polar products, most notably 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetraols). This implies the intermediacy of 7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydro-benzo[a]pyrenes (BPDE). We report here the sulfite-dependent conversion of BP-7,8-diol to forms highly mutagenic to Salmonella typhimurium strain TA 98. This activation is observed at BP-7,8-diol concentrations of from 2 to 40 microM and at sulfite concentrations of from 0.5 to 10 mM. In the presence of 10 microM BP-7,8-diol, half-maximal activation is observed at 1.6 mM sulfite. Sulfite itself is neither toxic nor mutagenic to the bacteria under these conditions. The time course of the activation of BP-7,8-diol and its sensitivity to inhibition by antioxidants indicate a requirement for sulfite autoxidation. These data further support the sulfite-dependent epoxidation of BP-7,8-diol. Not only does sulfite convert this promutagen to its active mutagenic form, sulfite also enhances the mutagenic activity of BP diolepoxides toward the tester strain. The reversion frequency in response to 0.1-0.5 microM anti-BPDE is increased by up to 33% in the presence of 1 mM sulfite, and by up to 270% with 10 mM sulfite. The mechanism of this enhancement of anti-BPDE activity is not known, but could be related to inhibition of the glutathione-S-transferase system which has been previously reported for sulfite. These results are discussed in regard to the noted cocarcinogenicity of sulfur dioxide for BP.

Peroxidatic oxidation of benzo[a]pyrene and prostaglandin biosynthesis.

The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.

Metabolism of arachidonic acid by hamster trachea lack of stimulation by A23187.

The metabolism of arachidonic acid has been studied using hamster trachea in short-term organ culture. To study endogenous substrate utilization, tissue lipids were labeled with [3H]-arachidonic acid, whereas exogenous substrate turnover was assessed by the addition of 100 microM [14C]-arachidonic acid to the medium. Both exogenous and endogenous arachidonate were converted primarily to 6-keto-PGF1 alpha and PGE2, with varying amounts of an unidentified non-polar product noted. Production of the prostanoids increased steadily with time up to 24 hours. No significant generation of lipoxygenase products was found. Release of incorporated labeled arachidonic acid was nearly linear with time, resulting in the transfer of about 10% of the total label into the medium after 24 hours. About 3% of the total label was converted to prostaglandins. In the presence of 10 microM A23187, release of label was increased by only 25 to 60% relative to the control. Analysis of labeled compounds in the medium showed that this increase resulted from increased release of unchanged arachidonic acid, and that the yield of oxygenated products was the same as from the control incubations.

Characterization of (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate.

The genotoxicity of certain benzo[a]pyrene (BP) derivatives is significantly enhanced in strains of Salmonella typhimurium following addition of sulfite to the incubations. The interaction between sulfite and those BP derivatives also results in the formation of isomeric BP sulfonates. As these trihydroxy sulfonates are formed in incubations of BP derivatives and sulfite in which a marked potentiation of bacterial mutagenicity occurs, we have investigated the properties of these novel intermediates. The compound (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate (BPT-9-sulfonate) was isolated and characterized in terms of its chemical and biological activity. This BPT sulfonate isomer is formed by the addition of the sulfite anion radical to the 9,10-double bond of the known promutagen, (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Evidence for the free radical character of this addition includes the initiation of the reaction by either peroxidase-catalyzed or chemical one-electron oxidation of sulfite, the inhibition of the reaction by phenolic antioxidants, and the isolation and characterization of the chain termination product, 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9,10-disulfonate (BPD-disulfonate). Analysis of incubations of S. typhimurium strain TA98 with BP-7,8-diol and sulfite, which resulted in a 10-fold increase in revertant bacterial colonies above control levels, showed that BPT-9-sulfonate and BPD-disulfonate were the only isolable products derived from BP-7,8-diol. This prompted a further investigation of the chemistry of these products. BPT-9-sulfonate was found to be quite stable in aqueous media, being refractory to acid- or base-catalyzed hydrolysis over a pH range of 3-11.(ABSTRACT TRUNCATED AT 250 WORDS)

Predominant 4-hydroxylation of estradiol by constitutive cytochrome P450s in the female ACI rat liver.

The ACI rat is extremely sensitive to estrogens as mammary carcinogens, whereas the Sprague-Dawley strain is relatively resistant. Comparison of the disposition and effects of estrogens in these two strains should provide insights into the mechanisms of estrogen carcinogenicity. We have begun this investigation by comparing the metabolism of [(3)H]17beta-estradiol (E2) by liver microsomes prepared from female rats from each strain. Both strains produce estrone (E1) as the major product at E2 concentrations >1 microM, with smaller amounts of 2-hydroxy-E2 formed. As the E2 concentration is decreased, however, aromatic hydroxylation becomes a more dominant pathway for both strains. At starting E2 concentrations as low as 3 nM, Sprague-Dawley liver microsomes produced comparable yields of 2-hydroxy-E2 and E1. In contrast, ACI liver microsomes yielded a profound shift to aromatic hydroxylation as the dominant pathway as E2 concentrations dropped below 1 microM, and this shift reflected the production of 4-hydroxy-E2 as the predominant product. The apparent K(m) for 4-hydroxylation of E2 is <0.8 microM, as opposed to approximately 4 microM for 2-hydroxylation, suggesting that different cytochrome P450s (CYPs) are responsible. Western immunoblotting of the liver microsomal preparations from ACI and Sprague-Dawley rats for CYPs known to catalyze 2- and 4-hydroxylation of E2 revealed that both strains contained comparable amounts of CYP 2B1/2 and 3A1/2, but no detectable amounts of CYP 1B1, the proposed E2 4-hydroxylase. Although this enzyme is not a constitutive CYP in Sprague-Dawley rat liver, its presence in ACI liver could provide a ready explanation for the predominance of 4-hydroxy-E2 as a product. The identity of the estradiol 4-hydroxylase in ACI rat liver and the role of this unique reaction in the heightened sensitivity to E2 carcinogenicity remain to be elucidated.

Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system.

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogen for benzo[a]pyrene (BP). We have demonstrated previously that the interaction between sulfite, the physiological form of sulfur dioxide, and (+/-) -7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenic form of BP, results in an enhanced mutagenic effect in Salmonella typhimurium strains TA98 and TA100. We report here that this same co-mutagenic effect of sulfite occurs in a mammalian cell line. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE, a concentration on the linear portion of the dose-response, resulted in a four-fold increase in mutations at the hprt locus relative to the spontaneous rate. When V79 cells were exposed to 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE, the mutation rate increased by 73% and 210%, respectively, over that elicited by anti-BPDE alone. Sulfite itself was moderately cytotoxic, but caused no increase in mutation over the spontaneous rate. Characterization of the dose- and time-dependance of this enhancement of diol epoxide mutagenicity by sulfite closely resembled the effects seen previously in the bacterial system. In particular, enhancement by sulfite was evident when sulfite was added to the cells between 60 min and 1 min prior to the addition of the diol epoxide. Concurrent addition of sulfite and the diol epoxide attenuated the enhancement, and the effect was lost altogether when sulfite was added 10 min after the diol epoxide. The specificity of this effect of sulfite was shown by comparison with sulfate, which at concentrations of either 1 or 10 mM exhibited modest cytotoxicity, but neither was directly mutagenic nor able to enhance the mutagenic effect of anti-BPDE. Binding studies with labeled anti-BPDE showed that the addition of 10 mM sulfite increased binding of anti-BPDE to DNA by over 43%, corresponding to the observed increase in mutant frequency. Interestingly, this difference in level of DNA modification was not apparent after 30 min to 2 h exposures, but only emerged at the 4 h time point. The 4 h point was routinely used for all mutagenicity studies. Binding of anti-BPDE-derived materials to cellular RNA was not altered by 10 mM sulfite. The emergence of increased DNA modification at the latest time point suggests either a more prolonged period of active DNA binding than would occur with diol epoxide, or a difference in the ability to recognize and clear specific DNA adducts. Both possibilities are discussed in regard to the observed formation of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a] pyrene-10c-sulfonate (BPT-10-sulfonate) in those incubations. BPT-10-sulfonate is a relatively stable BP derivative which retains the ability to covalently modify DNA. The role of this derivative in the enhancement of diol epoxide mutagenicity by sulfite is strongly suggested by these data.

Metabolism and activation of 7,8-dihydrobenzo[a]pyrene during prostaglandin biosynthesis. Intermediacy of a bay-region epoxide.

A Tween 20-solubilized preparation of prostaglandin endoperoxide synthase has been shown to metabolize 7,8-dihydrobenzo[a]pyrene (H2BP) to a form highly mutagenic to Salmonella typhimurium strain TA98. The arachidonic acid-dependent metabolism of H2BP by microsomal and purified prostaglandin endoperoxide synthase has been studied and the products identified. A spectral investigation of the metabolism indicated the bay-region double bond as the primary site of metabolism. Radiolabeled H2BP was synthesized and incubated with the enzyme preparations and the metabolites were separated by reverse phase high performance liquid chromatography and quantitated by liquid scintillation counting. Radioactive products were characterized by co-chromatography with chemically synthesized standards, UV-visible spectra, and mass spectrometry of acetate derivatives. The major polar products were determined to be trans- and cis-9,10-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8,9,10-tetrahydrobenzo[a]pyrene-9-one in a ratio of 1:1.2:0.4. The inclusion of 5 mM 3,3,3-trichloropropene-1,2-oxide, an epoxide hydrolase inhibitor, produced the same products but in a ratio of 1:2.3:1.2. Incubations with purified prostaglandin endoperoxide synthase yielded the three products in a ratio of 1:2.8:0.7. The major nonpolar product was identified as benzo[a]pyrene. The polar products of metabolism, the effects of 3,3,3-trichloropropene-1,2-oxide on their distribution, and the detection of a mutagenic intermediate support the conclusion that H2BP is co-oxygenated during prostaglandin biosynthesis to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.