Predicting carcinogenicity of petroleum distillation fractions using a modified Salmonella mutagenicity assay

The Ames Salmonella/microsomal activation mutagenesis assay has been modified to improve sensitivity and reproducibility to complex mixtures derived from the refining and processing of petroleum. Oil samples were dissolved in cyclohexane and subsequently extracted with dimethyl sulfoxide to produce aqueous compatible solutions which readily interact with tester bacteria. Also, the liver homogenate (S-9) and NADP cofactor concentrations were increased and hamster rather than rat liver S-9 was used. The initial slope of the dose response curve relating mutagenicity (revertants per plate) to the dose of extract added was used as an index of mutagenic activity, this slope was obtained through a computerized curve fitting procedure. The modified assay was used to rank 18 oil samples for mutagenic activity, this ranking correlates highly (r = 0.92) with potency rankings of the same samples previously determined from dermal carcinogenicity bioassays. Sensitivity and reproducibility of the assay are sufficient to permit routine use for detecting potential carcinogenic activity of individual refinery streams and blends which contain components boiling above 500°F.Abbreviations API American Petroleum Institute - B[a]P benzo[a]pyrene - DMSO dimethyl sulfoxide - NADP nicotinamide adenine dinucleotide phosphate - PAH polycyclic aromatic hydrocarbon - S-9 microsomal fraction from rat liver     

Test systems for biomonitoring based on membrane-bound enzyme complexes. IV. Assessment of genotoxic effects in an Ames test system with metabolic activation by the microsomal mono-oxygenases from fish livers

The study of mutagenic effect of 2-aminoantracene and benz(alpha)pyrene on Salmonella triphimurium TA 100 in the Ames test-system in the presence of postmitochondrial fractions S-9 from carp liver with 3-methylcholantrene induced by microsomal oxidation system has been carried out. The metabolic activity and cytochrome P450 contence in carp liver microsomes have been shown to concede considerably those in rats liver. But these characteristics are sufficient for the use of fraction S-9 from carp liver for the study of genotoxic effect of these xenobiotics in the Ames test-system. Several regimes of storage of S-9 preparations from carp liver have been compared. S-9 preparations frozen immediately after isolation preserve their metabolic activity with respect to 2-aminoantracene and benz(alpha)pyrene well.     

Inhibitory effect of organic solvents on the mutagenicity of N-nitrosodialkylamines in Salmonella

The influence of organic solvents on the mutagenicity of 11 N-nitrosamines was examined in Salmonella typhimurium TA100 using the Ames's liquid incubation assay in the presence of rat-liver S9. The mutagenic activities of N-nitrosodimethylamine, N-nitrosodiethylamine, 6 oxidative derivatives of N-nitrosopropylamine and N-nitroso-2,6-dimethylmorpholine were considerably decreased by addition of dimethyl sulfoxide, dimethyl formamide, acetone, 95% ethanol or acetonitrile, which are recommended for use as solvents in the assay by Ames's group, to the incubation mixture. The mutagenic activities of N-nitrosodipropylamine and N-nitrosodibutylamine, which are barely soluble in water, were also suppressed by increasing concentrations of dimethyl sulfoxide. These organic solvents did not appear to exert their influence by desmutagenic and antimutagenic actions. In contrast, the recoveries of unmetabolized carcinogens from preincubation mixtures and from agar plates were significantly higher in the presence of organic solvents than in their absence. The results indicate that the inhibitory effect is a result of interference with the process of metabolic activation by liver S9.     

The genetic toxicity of 1,2-dibromo-3-chloropropane, 1,2-dibromo-3- chloro-2-methylpropane,and 1,2,3-tribromo-2-methylpropane

1,2-Dibromo-3-chloro-2-methylpropane (DBCMP) and 1,2,3- tribromo-2-methylpropane (TBMP) are contaminants formed during the manufacture of bromobutyl rubber. These chemicals are structurally similar to 1,2-dibromo-3-chloropropane (DBCP), a known genotoxin and rodent carcinogen. The present study compared the genotoxic properties of DBCMP and TBMP to those of DBCP. In the Salmonella assay, DBCP was positive in strains TA98, TA-100 and TA-1535 in the presence of exogenous activation; DBCP was weakly active in TA-1535 in the absence of activation. Neither DBCMP nor TBMP produced reproducible evidence of mutagenic activity in the Salmonella assay despite the use of several different variations of this test. In the mouse lymphoma gene mutation assay DBCP and TBMP were positive in the presence and absence of activation, while DBCMP was positive only in the absence of activation. All three test compounds were active in the Syrian hamster embryo morphologic transformation assay. The results indicated that both DBCMP and TBMP exhibited some genotoxic activity as did DBCP. The presence of the methyl group on the 2-carbon position essentially eliminated the mutagenicity of DBCMP and TBMP in the Salmonella assay.abbreviations CHO Chinese hamster ovary cells - DBCMP 1,2-dibromo-3-chloro-2-methylpropane - DBCP 1,2-dibromo-3-chloropropane - DMEM Dulbecco's Eagle's minimal E medium - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - S-9 microsomal fraction from rodent liver - TBMP 1,2,3-tribromo-2-methylpropane - TBP 1,2,3-tribromopropane - TFT trifluorothymidine     

Resorufin inhibits the in vitro metabolism and mutagenesis of benzo(a)pyrene

7-Hydroxyphenoxazin-3-one, commonly known as resorufin, strongly inhibits benzo(a)pyrene-induced mutation in the Ames bacterial reversion assay. The antimutagenic mechanism is due in part to redox cycling of resorufin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen. The diversion of electrons from cytochrome P-450 enzymes results in a large decrease in the percent of benzo(a)pyrene metabolized by rat liver microsomes as measured by HPLC. Resorufin stimulated a non-stoichiometric consumption of NADPH and was reduced in S-9 or microsomal solutions. These processes were sensitive to dicumarol and NADP inhibition to different degrees in each liver fraction. This suggests two pathways are involved in resorufin redox cycling, one involving DT-diaphorase and the other with NADPH cytochrome P-450 reductase. Oxygen was shown to be an electron acceptor for S-9 mediated resorufin redox cycling, but was not consumed by a microsomal solution in the presence of resorufin and NADPH.     

The process of promutagen biotransformation studied by the Ames test. II. The extent of the manifestation of the mutagenic action of nitrosomorpholine, diethylnitrosamine and cyclophosphane using various subfractions of rat liver homogenate

The data are presented on involvement of components of microsomal and cytosolic subfractions composing the S-9 fraction of rat liver homogenate in processes leading to formation of active metabolites of nitrosomorpholine (NM), diethyl nitrosoamine (DENA) and cyclophosphane (CP) promutagens and their detoxication resulting from the reaction with glutathione (G-SH) added to the system. It is established that the process of metabolic activation is only connected with microsomal subfraction, while reactions of the first phase of CP and DENA metabolism take place when both microsomal and cytosolic subfractions are added. Decrease in the effect of all promutagens studied under the action of G-SH was observed after microsomal and cytosolic subfractions of the S-9 fraction were introduced into the activating mixture. Various values of dependence of the metabolic activation level and the extent of decrease in the mutagenic action, upon addition of G-SH, on the protein content in microsomal and cytosolic subfractions were obtained.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

Dimethylnitrosamine metabolism: I. In vitro activation of dimethylnitrosamine to mutagenic substance(s) by hepatic and renal tissues from three inbred strains of mice

The potential of hepatic and renal homogenates from three inbred strains of mice (BALB/c, C57BL and DBA) to activate dimethylnitrosamine (DMN) was investigated. Microsomal enzyme (S-9) preparations of liver and kidney from mature and immature mice were used in the Ames Salmonella mutagenicity assay. No age or sex-related differences in the formation of active mutagenic DMN Metabolites by liver microsomal enzymes were observed within any of the three inbred strains. In contrast, mature male kidney S-9 fractions from all three strains had a significantly greater potential to activate DMN than mature female and immature animals. Testosterone treatment resulted in no apparent changes in the ability of hepatic tissue to biotransform DMN to its mutagenic metabolites among age and sex classes. However, after testosterone treatment, renal microsomal fractions from mature female mice of all three strains did not differ significantly from their male counterparts in their ability to transform DMN to mutagenic metabolites.     

Determination of the characteristics, properties and homogeneity of rat brain microsomes. Binding of lactate dehydrogenase, malate dehydrogenase and 5' nucleotidase to microsomal membranes

Quantitatively, the amount of microsomes obtained using dimethyl sulfoxide is larger than that obtained from sucrose solutions (Centelles, Franco & Bozal (1986) Biol. Chem. Hoppe Seyler 367, 461-475). In this paper it is demonstrated that from a qualitative point of view they appeared to be indistinguishable with respect to molecular characteristics. Thus, both types of microsomes had the same behaviour in experiments of isopicnic ultracentrifugation with Percoll, isoelectric focusing and gel permeation. In these experiments, the 5'-nucleotidase, lactate dehydrogenase and malate dehydrogenase activities bound to the microsomal fraction were also studied. Lactate and malate dehydrogenase activities were always found in free and membrane-bound form. In contrast, 5'-nucleotidase activity was always encountered bound to microsomal membranes.     

Ascorbyl free radical and dehydroascorbate formation in rat liver endoplasmic reticulum

The mechanism of ascorbate oxidation was studied in rat liver microsomes. A continuous consumption of the added ascorbate was observed, which was accompanied with a prompt appearance of ascorbyl free radical and dehydroascorbate. Microsomes sustained steady-state level of ascorbyl free radical and dehydroascorbate till ascorbate was present in the medium. Ascorbyl free radical formation was diminished when microsomes had been pretreated with heat or trypsine. It was also decreased by addition of quercetin, econazole or metal chelators, including the copper specific neocuproine. Enzymatic (superoxide dismutase, catalase) and nonenzymatic (dimethyl sulfoxide, mannitol) antioxidants did not modify the microsomal production of ascorbyl free radical. Investigation of the subcellular distribution of ascorbate oxidation showed that the microsomal fraction of liver had the highest activity. The decrease of ascorbate oxidation after protease treatment and the negligible increase upon permeabilization of microsomal vesicles showed that a membrane protein is responsible for the activity, which is exposed to the outer surface of the endoplasmic reticulum. The results indicate the presence of a primary enzymatic ascorbate oxidation in rat liver endoplasmic reticulum which is able to generate dehydroascorbate, an important source of the oxidizing environment in the endoplasmic reticulum.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems.

A pathway for the synthesis of dimethyl selenide from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30,000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO2 (93% inhibition by 10(-6) M arsenite in the presence of a 20,000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction

Chlorotrifluoroethene, a potent nephrotoxin, is a substrate for the glutathione S-transferases present in the cytosolic and microsomal fractions of rat liver. The glutathione conjugate formed by both subcellular fractions has been identified as S-(2-chloro-1,1,2-trifluoroethyl)glutathione by 1H and 19F NMR and by secondary ion mass spectrometry. The conjugate formed by the cytosolic fraction is an equimolar mixture of two diastereomers, whereas the conjugate formed by the microsomal fraction is predominantly one diastereomer, as judged by the 19F NMR spectra. No evidence for the formation of S-(trihalovinyl)glutathione derivatives by an addition/elimination reaction was found. High-performance liquid chromatography was employed to measure the rates of glutathione conjugate formation in vitro. The rates of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation were 75-107 nmol min-1 (mg of protein)-1 and 151-200 nmol min-1 (mg of protein)-1 catalyzed by the cytosolic and microsomal fractions, respectively (measured at pH 7.4, 37 degrees C, with 5 mM glutathione). These results suggest that glutathione conjugation occurs at high rates in vivo to produce the highly nephrotoxic S-(2-chloro-1,1,2-trifluoroethyl)glutathione.     

UDP-galactose:glycoprotein galactosyltransferase activity in tissues of developing rat

UDP-galactose:glycoprotein galactosyltransferase activity has been measured in several tissues of the rat, ranging in age from 16 days embryo to 35 days postnatal. The enzyme activity was found to be high in fetal liver, lungs, and brain tissues but the concentration decreased with gestational age with no further changes after birth. The enzyme activity in the serum of newborns was higher than in pregnant and nonpregnant adult rats. There was no qualitative difference (optimum pH, cation requirements, affinity for the substrate UDP-galactose, or requirement for Triton X-100) between the enzyme from embryonic liver and that from adult rats. During the embryonic stage nearly half of the enzyme activity was localized in a plasma membrane-rich fraction and only a minor part in the microsomal fraction, while in the adult most of the activity was present in the microsomal fraction. Under certain conditions of assay the incorporation of galactose into glycoprotein in liver homogenates was greatly stimulated by CDP-choline or ATP. However, CDP-choline showed a considerably greater effect than ATP at 5 days after birth but this effect could be eliminated by solubilizing the homogenates in deoxycholate.     

Detection of mutagenicity in cervico-vaginal secretions--a plausible risk factor for cervical cancer

The cervico-vaginal secretions from 51 women with various grades of dysplastic lesions of uterine cervix were assessed for mutagenic potential by Ames test using histidine deficient mutant strain of Salmonella typhimurium TA-98: with S-9 mix. Twenty three per cent of samples from women with cervical dysplasia were found significantly positive (P less than 0.001) for mutagenic activity compared to 3% positive from control. The frequency of mutagenic secretions detected were almost uniform, irrespective of the severity of cervical lesions. None of cervico-vaginal secretions, positive for mutagenicity could revert the tester strain when tested in absence of S-9 mix (liver microsomal enzymes). This indicates that mutagens in cervico-vaginal secretions are effective only when activated enzymatically.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems

A pathway for the synthesis of dimethyl seledine from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30 000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO₂ (93% inhibition by 10^?6 M arsenite in the presence of a 20 000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.     

Genotoxicity of diesel-exhaust particles dispersed in simulated pulmonary surfactant.

Diesel-exhaust particles from two sources were dispersed in aqueous mixtures of dipalmitoyl phosphatidyl choline, a major component of pulmonary surfactant, and were tested for genotoxicity. Diesel samples from the same sources were extracted with dichloromethane and transferred into dimethyl sulfoxide and subjected to the same assays. Both types of extractions yielded similar results in both the Salmonella mutagenicity assay and the sister-chromatid exchange assay using V79 cells. After separation of the samples into supernatant and sediment fractions, the activity of both diesel samples was shown to reside exclusively in the supernatant fraction for the solvent-extracted samples, and exclusively in the sedimented fraction for surfactant dispersed samples. These findings indicate that genotoxic activity associated with diesel particles inhaled into the lung may be made bioavailable by virtue of the solubilization/dispersion properties of pulmonary surfactant components.     

Pyrazole and 4-methylpyrazole inhibit oxidation of ethanol and dimethyl sulfoxide by hydroxyl radicals generated from ascorbate, xanthine oxidase, and rat liver microsomes

Pyrazole and 4-methylpyrazole, which are potent inhibitors of alcohol dehydrogenase, inhibited the oxidation of ethanol and of dimethyl sulfoxide by two model hydroxyl radical-generating systems. The systems used were the iron-catalyzed oxidation of ascorbic acid and the coupled oxidation of xanthine by xanthine oxidase. Pyrazole and 4-methylpyrazole were more effective inhibitors at lower substrate concentrations than at higher substrate concentrations; the oxidation of ethanol was inhibited to a greater extent than the oxidation of dimethyl sulfoxide. These results are consistent with competition between pyrazole or 4-methylpyrazole with the substrates for the generated hydroxyl radicals. Pyrazole and 4-methylpyrazole appear to be equally effective in reacting with hydroxyl radicals. An approximate rate constant of about 8 × 10⁹m^?1 s^?1 was calculated from the inhibition curves, indicating that pyrazole and 4-methylpyrazole are potent scavengers of the hydroxyl radical. Previous studies have implicated a role for hydroxyl radicals in the microsomal pathway of ethanol oxidation. In the presence of azide (to inhibit catalase), pyrazole and 4-methylpyrazole inhibited the NADPH-dependent microsomal oxidation of ethanol, as well as several other hydroxyl radical-scavenging agents. This inhibition by pyrazole and by 4-methylpyrazole may reflect a mechanism involving competition for hydroxyl radicals generated by the microsomes. However, the kinetics of inhibition by pyrazole were mixed, not competitive, and pyrazole and 4-methylpyrazole also inhibited aminopyrine demethylase activity. Pyrazole has been shown by others to interact with cytochrome P-450. It is suggested that pyrazole and 4-methylpyrazole affect microsomal oxidation of ethanol via effects on the mixed-function oxidase system and via competition for the generated hydroxyl radicals. In view of these results, low concentrations of pyrazole and 4-methylpyrazole should be used in studies on pathways of alcohol metabolism, and caution should be made in interpreting the actions of these compounds when used at high concentrations.     

Guinea pig liver aldehyde oxidase as a sulfoxide reductase: Its purification and characterization

Guinea pig aldehyde oxidase was purified about 120-fold at a yield of 26% from liver cytosol by sequential column chromatography using DEAE-cellulose, FMN-Sepharose 4B, and Sephacryl S-300. The purified enzyme showed many similarities with the rabbit liver aldehyde oxidase reported by other workers with respect to its absolute spectra, molecular weight, and cofactor compositions of molybdenum, FAD, and nonheme iron. This enzyme efficiently utilized 2-hydroxypyrimidine and benzaldehyde as electron donors while N¹-methylnicotinamide was 40 times less effective than 2-hydroxypyrimidine. Diphenyl sulfoxide was reduced anaerobically to diphenyl sulfide in the presence of electron donors. This activity was highly susceptible to SKF 525-A as well as the known inhibitors for aldehyde oxidase such as menadione, estradiol, and potassium cyanide. This enzyme also reduced dibenzyl sulfoxide, phenothiazine sulfoxide, d-biotin methyl ester d-sulfoxide, and quinoline N-oxide, but not l-methionine sulfoxide, dimethyl sulfoxide, d-biotin methyl ester l-sulfoxide, and d-biotin d- and l-sulfoxides, as well as diphenyl sulfone. These results indicate that aldehyde oxidase in guinea pig liver functions as a sulfoxide reductase with selective substrate specificity under anaerobic conditions.