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G A Reed

Publications and source records attributed to G A Reed.

At least 19 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

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.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

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.

Administration, Topical↗
PubMed1994

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)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

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.

Biotransformation↗
PubMed1991

Mutagenicity of benzo[a]pyrene bay-region sulfonates.

The interaction between the sulfite anion and specific benzo[a]pyrene (B[a]P) derivatives produces a novel class of benzo[a]pyrene sulfonates. (+/-)-7,8,9-Trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-sulfonate (B[a]PT-10-sulfonate) is formed in high yields in incubations containing (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (anti-BPDE) and sulfite, and sulfite strongly enhances the mutagenicity of the diolepoxide toward Salmonella typhimurium under those conditions. Although B[a]PT-10-sulfonate itself shows little direct mutagenicity over a 1-20 microM concentration range, this reactive bay-region intermediate does enhance the mutagenicity of anti-BPDE in strains TA98 and TA100 by up to 280%. No significant enhancement was seen when up to 20 microM B[a]PT-10-sulfonate was used in concert with another direct-acting mutagen, N-acetoxy-acetylaminofluorene (N-AcO-AAF). The isomeric product derived from sulfite and (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) is (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate (B[a]PT-9-sulfonate). Like B[a]PT-10-sulfonate, B[a]PT-9-sulfonate is not mutagenic to strains TA97, TA98 and TA100. This sulfonate exhibited little enhancing activity with anti-BPDE over a 1-20 microM concentration range, but did enhance the mutagenic response of strain TA98 to 0.2 microM N-Aco-AAF by up to 128%. Sulfite, anti-BPDE and B[a]PT-sulfonates were also examined for the ability to induce a forward mutation at the hgprt locus (8-azaguanine resistance) in strains of S.typhimurium. Sulfite caused a marked enhancement of forward mutation due to anti-BPDE in both TA98 and TA100. Surprisingly, concurrent administration of B[a]PT-10-sulfonate with anti-BPDE did not increase the number of mutant colonies. The extensive conversion of anti-BPDE to B[a]PT-10-sulfonate under conditions where sulfite enhances diolepoxide mutagenicity, when coupled with this enhancement of diolepoxide mutagenicity by B[a]PT-10-sulfonate in the reverse mutation assay, supports this novel B[a]P derivative as a mediator of the sulfite-dependent enhancement of B[a]P genotoxicity. Determining why this enhancing effect was not seen when selecting for mutation at the hgprt locus of S.typhimurium will require further study.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

Peroxyl radical-dependent epoxidation of cyclopenteno[c,d]pyrene.

We have reported previously that cyclopenteno[c,d]pyrene (CPP), an environmentally prevalent polycyclic aromatic hydrocarbon, is activated as a bacterial mutagen by several model systems which generate peroxyl radicals. In this report we present our findings on the chemical fate of CPP in these activating systems. The peroxyl radical systems employed are microsomal prostaglandin H synthase and arachidonic acid, the hematin-catalyzed decomposition of a lipid hydroperoxide, and the autoxidation of the sulfite anion. Reverse-phase HPLC analysis of stable products of CPP metabolism yielded qualitatively identical profiles from the first two systems. The three major products from these systems were analyzed by UV/visible and fluorescence spectroscopy, and a mass spectrum was obtained for the most abundant product. Based on these spectral analyses and on chromatographic behavior, the three products were identified as the cis- and trans-isomers of 3,4-dihydroxy-3,4-dihydro-CPP and 4-keto-(3H)-CPP. The identities of these products and their quantitative distributions relative to the epoxide hydrolase activities present in the microsomal system and the hematin system clearly establish 3,4-epoxy-CPP as the key intermediate and probable active mutagen generated in the peroxyl radical-dependent metabolism of CPP. This epoxidation of the activated aliphatic double bond of CPP extends the known range of peroxyl radical-dependent oxygenations by demonstrating the direct, one-step activation of a carcinogenic, environmentally relevant hydrocarbon. Strikingly different results are obtained in the sulfite-dependent system. The epoxide-derived metabolites seen with the peroxyl radical systems are very minor products. Instead, two product peaks elute near the solvent fron on reverse-phase HPLC. These are apparently monohydroxy-CPP sulfonates. Such products may form either by the direct addition of the sulfite anion radical to the activated double bond of CPP or by peroxyl radical-dependent epoxidation of CPP followed by nucleophilic addition of sulfite. Precedent for both of these reactions has been reported with analogous benzo[a]pyrene derivatives. The occurrence of these radical-dependent transformations in intact mammalian systems has not been investigated, but the ability of all three model systems employed to convert CPP to potent bacterial mutagens implies that these pathways should be studied further.

Animals↗
PubMed1990

Sulfite enhancement of diolepoxide mutagenicity: the role of altered glutathione metabolism.

Sulfur dioxide is a cocarcinogen for benzo[a]pyrene in the respiratory tract of rats and hamsters. Sulfur dioxide exists under physiological conditions as the sulfite ion. Sulfite enhances the mutagenic potency of (+-)-7r,8t-dihydroxy-9t,-10t- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) and 7r,8t-dihydroxy-9c10c-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e (syn-BPDE) in Salmonella typhimurium strains TA98 and TA100. This enhancement of diolepoxide mutagenicity is observed with sulfite concentrations between 1 and 20 mM, and the concentration dependence is identical for the two diolepoxides. Half-maximal enhancement of mutagenicity occurs at approximately 5 mM sulfite. Sulfite is neither toxic nor mutagenic to the bacteria under these conditions. The enhancement of diolepoxide mutagenicity requires that the bacteria be exposed to sulfite prior to the addition of the diolepoxide. Simultaneous addition of sulfite and diolepoxide significantly decreases the enhancing effect, and addition 15 min after the diolepoxide virtually abolishes the effect. This is consistent with sulfite serving to increase the efficiency of processes leading to DNA modification by the diolepoxides, rather than some effect subsequent to DNA adduct formation. Direct evidence for this hypothesis was provided by determining the effect of sulfite on mutagenicity and DNA binding in TA98 using [3H]anti-BPDE. Exposure of the bacteria to 10 mM sulfite for 5 min prior to the addition of the labeled mutagen led to as much as 170% increase in DNA binding levels relative to parallel incubations without sulfite. Corresponding increases in mutagenicity were seen as well. As sulfite can affect the glutathione/glutathione-S-transferase systems, the primary cellular defense against BPDE, the effect of sulfite on these pathways in Salmonella was determined. When strain TA98 was treated with N-acetoxy-2-acetamidofluorene, a direct-acting mutagen not scavenged by glutathione, prior addition of 10 mM sulfite to the bacteria had no effect on resultant viability or mutagenicity. Assessment of the bacterial glutathione levels revealed that 10 mM sulfite treatment results in an 82% decrease in the concentration of the cosubstrate. We were, however, unable to detect diolepoxide-glutathione conjugates in any of our incubations. Moreover, the presence of sulfite leads to significant trapping of the diolepoxide in the form of sulfonate derivatives. Based on these data, we conclude that the depletion of glutathione does indeed play a role in the enhancement of diolepoxide mutagenicity in S. typhimurium.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase.

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Metabolic activation of cyclopenteno[c,d]pyrene by peroxyl radicals.

The conversion of cyclopento[c,d]pyrene (CPP) to forms which are mutagenic to Salmonella typhimurium strain TA98 has been demonstrated in systems which generate peroxyl radicals. The systems examined included prostaglandin H synthase (PHS) and arachidonic acid, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) and hematin, and the autoxidation of the sulfite ion. In all cases concentration-dependent activation of CPP was observed at hydrocarbon concentrations between 10 and 100 microM. Neither CPP nor the peroxyl radical systems alone were mutagenic or toxic to the tester strain. The use of hydroxygen peroxide with PHS, a peroxidative system which does not yield peroxyl radicals, does not activate CPP. The involvement of a CPP epoxide was examined using 1,1,1-trichloropropene-2,3-oxide. Addition of this epoxide hydrolase inhibitor to incubations of CPP with the PHS/arachidonic acid system resulted in a 210% increase in induced revertants relative to the system in the absence of the inhibitor. The addition of pure rat liver microsomal epoxide hydrolase to incubations of CPP with the 15-HPETE/hematin system resulted in a concentration-dependent loss of mutagenicity, further supporting the intermediacy of an epoxide. The site of metabolism of CPP is the cyclopenteno double bond based on the formation of products which display distinct pyrene-type fluorescence spectra. The involvement of the cyclopenteno double bond also is shown by the inability of the 15-HPETE/hematin system to activate 3,4-dihydrocyclopenteno[c,d]pyrene as a mutagen. CPP is the first environmentally-relevant carcinogenic hydrocarbon found to be activated directly by peroxyl radical systems without prior biotransformation to a diol derivative by the cytochrome P-450 system. These findings expand the range of potentially toxic substrates to be considered for activation by peroxyl radical pathways.

Animals↗
PubMed1987

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.

Benzo(a)pyrene↗
PubMed1986

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.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Epoxidation of (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene during (bi)sulfite autoxidation: activation of a procarcinogen by a cocarcinogen.

The (bi)sulfite ion undergoes extensive autoxidation in neutral aqueous media with the formation of sulfur trioxide radical anion that is detected by ESR. The radical anion subsequently reacts with molecular oxygen to form a peroxyl radical. We find that when (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is included in this autoxidation system, BP-7,8-diol is converted to diolepoxides, ultimate carcinogenic derivatives of benzo[a]pyrene. This epoxidation occurs with a stereoselectivity consistent with either a peroxyl radical or a peracid as the epoxidizing agent. The epoxidation is dependent on the concentration of both (bi)sulfite and oxygen. In the presence of 10 microM butylated hydroxyanisole, which abolishes (bi)sulfite autoxidation, no (bi)sulfite-dependent epoxidation occurs. These results are discussed in regard to the mechanism of (bi)sulfite autoxidation, and in relationship to the cocarcinogenicity of sulfur dioxide [anhydrous (bi)sulfite] for benzo[a]pyrene-induced pulmonary neoplasia.

Carcinogens↗
PubMed1985

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis.

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.

Animals↗
PubMed1985

Inactivation of prostaglandin H synthase and prostacyclin synthase by phenylbutazone. Requirement for peroxidative metabolism.

Phenylbutazone (PB), a nonsteroidal anti-inflammatory drug, is an efficient reducing cofactor for the peroxidase activity of prostaglandin H synthase (PHS). Most reducing cofactors for the peroxidase protect PHS and prostacyclin synthase from inactivation by hydroperoxides. PB, however, does not protect these enzymes, but rather augments their hydroperoxide-dependent inactivation. Using ram seminal vesicle microsomes as a source of PHS and prostacyclin synthase, we have examined the interaction of PB and exogenous hydroperoxides. Chromatographic analysis of the metabolism of 14C-labeled arachidonic acid in this system revealed that PB-dependent inactivation of PHS is markedly increased in the presence of 100 microM H2O2. This inactivation is a linear function of PB concentration between 10 and 250 microM, with a half-maximal effect in this range at about 100 microM PB. Prostacyclin synthase is even more sensitive to inactivation by the combined PB and H2O2 treatment, with a corresponding half-maximal effect at PB concentrations near 25 microM. This PB- and H2O2-dependent inactivation is demonstrable whether PGH2 is generated in situ from arachidonic acid or is added exogenously, supporting a direct effect of the treatment on prostacyclin synthase. As PB undergoes peroxide-dependent co-oxygenation catalyzed by PHS, we propose that it is an oxygenated derivative of PB, rather than the parent compound, which is responsible for the inactivation of PHS and prostacyclin synthase. Nafazatrom, a competitive inhibitor of PB co-oxygenation, blocks the effects of the PB and H2O2 treatment, supporting our proposal.

Animals↗
PubMed1984

Phenylbutazone-dependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. A new mechanism for prostaglandin H synthase-catalyzed oxidations.

The nonsteroidal anti-inflammatory drug phenylbutazone markedly enhances the hydroperoxide-dependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene catalyzed by microsomal and Tween-20 solubilized preparations of prostaglandin H synthase. Furthermore, phenylbutazone radically alters the hydroperoxide specificity of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene epoxidation. In the absence of phenylbutazone, only allylic hydroperoxides are effective in initiating epoxidation, whereas in the presence of phenylbutazone the reaction can be initiated by t-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide. All effects are dependent on the concentration of phenylbutazone present. The primary event is the oxidation of phenylbutazone by prostaglandin H synthase. This pathway yields a peroxy radical of phenylbutazone which appears to be the epoxidizing agent. This activation of a primary substrate by a peroxidase resulting in metabolism of a secondary substrate is analogous to the halogenation reactions catalyzed by chloroperoxidase. This represents a new class of oxidation reactions catalyzed by prostaglandin H synthase.

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