Biomedical subjects

P D Josephy

Publications and source records attributed to P D Josephy.

At least 37 records · Page 2Linked to original sources

PubMed1995

Bioactivation of aromatic amines by recombinant human cytochrome P4501A2 expressed in Ames tester strain bacteria: a substitute for activation by mammalian tissue preparations.

The most widely used bioassay in genetic toxicology is the Ames test, which combines a bacterial mutagenicity assay (reversion of Salmonella typhimurium histidine-auxotrophic tester strains) with an exogenous bioactivation system (hepatic postmitochondrial supernatant or "S9"). The enzymatic activities of S9 prepared from the tissues of experimental animals are difficult to control. We show that the requirement for S9 can be obviated by the engineered expression of enzymes of bioactivation within the bacterial cell. With this strategy, reactive metabolites are produced inside the bacterial cell, proximate to the genetic target. Species boundaries can be crossed, and chimeric or mutant enzymes can be studied. We have constructed an Ames tester strain, expressing both aromatic amine N-acetyltransferase and human cytochrome P4501A2, which detects aromatic amine mutagenicity in the absence of S9.

Acetyltransferases↗

PubMed1995

Metabolic activation of heterocyclic aromatic amines catalyzed by human arylamine N-acetyltransferase isozymes (NAT1 and NAT2) expressed in Salmonella typhimurium.

Heterocyclic aromatic amines formed during the cooking of meat and meat-derived products can be activated to reactive metabolites which bind to DNA, induce mutations and cause tumors in animals. A principal route of metabolic activation is N-oxidation to hydroxylamines, and their subsequent activation by acetyltransferase-catalyzed O-acetylation. We have used mutagenicity assays to study O-acetylation of heterocyclic arylhydroxylamines by the two isozymes of human N-acetyltransferase, NAT1 and NAT2, expressed in Salmonella typhimurium. N-Acetylation was also examined, using an HPLC method. In addition, Salmonella strains with endogenous acetyltransferase and lacking this activating activity were used. Hydroxylamines of nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx, NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situ by rat liver S9 mix. The strains expressing human NAT1 and lacking acetyltransferase activity showing little or no ability to activate these substrates. The strains expressing human NAT2 and Salmonella acetyltransferase supported to different extents the activation of all the compounds except PhIP and Trp-P-2. N-Acetylation of IQ, MeIQx and PhIP was slow or not detectable. In conclusion, human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines. NAT2 probably plays a key role in the genotoxic effects of the above heterocyclic amines except for PhIP and Trp-P-2, which have NAT2-independent mutagenic activity.

Acetylation↗

PubMed1995

Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and nitroaromatic compounds.

Escherichia coli lacZ strains CC107-CC111, which detect specific frameshift mutations, were used to study the mutational specificities of 2-nitro-3-methylimidazo[4,5-f] quinoline (NO2-IQ) and rat hepatic S9-activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). New constructs were made in which UvrABC-dependent excision repair was eliminated (strains DJ3107-DJ3111), followed by introduction of plasmid pYG219 conferring acetyl CoA:arylamine N-acetyltransferase/acetyl CoA:arylhydroxylamine O-acetyltransferase (NAT/OAT) activity (strains DJ3207-DJ3211). Sensitivity to mutagens was greatly enhanced. The mutational specificity of NO2-IQ was identical to that of the corresponding amine, IQ. The most prominent mutations caused by the two compounds were -2(CGGC) and 1(CG) frameshifts. +1(AT) Frameshifts play a minor role in the pattern of mutational specificity. Induction of all three mutations was similarly influenced by NAT/OAT activation and UvrABC-dependent excision repair. These new tester strains provide an effective tool for the study of aromatic amine mutational specificity and the influences of excision repair and NAT/OAT activation.

Acetyltransferases↗

PubMed1995

In vitro formation, disposition and toxicity of N-acetoxy-sulfamethoxazole, a potential mediator of sulfamethoxazole toxicity.

Variation in the formation and disposition of the hydroxylamine of (SMX-HA) is thought to play an important role in the pathogenesis of sulfamethoxazole (SMX)-induced idiosyncratic adverse drug reactions. We hypothesized that, in analogy to carcinogenic arylamines, SMX-HA might be further converted to an electrophilic N-acetoxy metabolite which could play a role in mediating SMX toxicity. Accordingly, we chemically synthesized N-acetoxy-SMX, and examined the characteristics of its formation, metabolism, cytotoxicity and mutagenicity in human and bacterial test systems. The human arylamine N-acetyl-transferases, (NAT)1 and NAT2, were capable of converting SMX-HA to N-acetoxy-SMX. NAT1 and NAT2 possessed similar affinities for SMX-HA (apparent Km values of 650 and 520 microM, respectively), but the apparent maximal velocity of the NAT1-mediated acetylation was higher than that of NAT2. (1332 vs. 37 nmol/min/U of immunoreactive NAT protein). Human peripheral blood mononuclear cells 12,000 x g supernatant fractions converted N-acetoxy-SMX mainly back to SMX-HA, and also to a lesser extent to SMX, at clinically relevant concentrations. Similar pathways were observed in human hepatic cytosolic fractions. In a cytotoxicity assay, N-acetoxy-SMX was significantly more toxic to human peripheral blood mononuclear cells than SMX-HA (16.6 vs. 11.5% dead cells at a concentration of 300 microM). N-acetoxy-SMX was weakly mutagenic to the Salmonella typhimurium TA100 strain in the Ames test. These data suggest that the N-acetoxy metabolites of sulfonamides could potentially play a role in mediating sulfonamide idiosyncratic adverse drug reactions.

Acetyltransferases↗

PubMed1994

Mutational spectrum of revertants in the hisD3052 allele of Salmonella typhimurium induced by hydrogen peroxide-activated benzidine.

Benzidine is mutagenic in a modified Ames (Salmonella typhimurium) assay, which uses hydrogen peroxide-dependent peroxidative activation. The mutational specificity of benzidine was investigated in tester strain TA98, which reverts by frameshifts of the hisD3052 allele. The most frequently observed mutation is a deletion of two bases from a (CG)4 run. This deletion was elevated in frequency among benzidine-induced revertants, relative to spontaneous revertants. Many other mutations were also observed, including additions, deletions, and complex events. Only small frameshifts were observed among the benzidine-induced revertants, whereas some larger deletions were observed among the spontaneous revertants.

Alcohol Oxidoreductases↗

PubMed1994

Dimethylnitrosamine genotoxicity: does N-acetyltransferase activity play a role?

Dimethylnitrosamine (DMN) genotoxicity was evaluated in two test systems: induction of the umu operon (as measured by expression of a lacZ gene fusion) and mutagenicity (as measured by the Ames assay). Three Salmonella typhimurium strains were used; the strains differ in the level of expression of the enzyme acetyl CoA:arylamine N-acetyltransferase (NAT). We did not observe increased sensitivity in a strain with a plasmid-borne copy of the nat gene, and expressing very high levels of NAT activity. In contrast to a recent report, we conclude that NAT-dependent metabolic activation does not play a major role in the genotoxicity of this carcinogenic nitrosamine.

Anthracenes↗

PubMed1994

Preliminary characterization of a factor in human follicular fluid that stimulates human spermatozoa motion.

Follicular fluid alters the physiology and behaviour of spermatozoa by increasing acrosome reaction, accelerating capacitation, attracting the spermatozoon and enhancing vigorous motion of the cell. The objective of this study was to characterize the factor(s) in human follicular fluid that causes vigorous spermatozoa motion. Follicular fluid and its fractions were tested for stimulation of spermatozoa motion using a standardized assay which employs a computerized digital imaging system. Our results show that both follicular fluid and its methanol extract stimulate vigorous spermatozoa motion. To determine the characteristics of the active factor(s), the methanol extract was subjected to molecular weight fractionation, protease digestion, microcrystalline thin-layer chromatography (TLC) and C18 reverse-phase high-pressure liquid chromatography (HPLC). The spermatozoa motion stimulator in the methanol extract was dialysable against a low molecular weight membrane (1000 Da), insensitive to boiling and low pH (3.5) and was largely inactivated by proteinase K digestion. The activity was detected near the solvent front on TLC. Using reverse-phase HPLC monitored at 254 nm (UV), the activity eluted as a single peak of activity at low methanol concentration, indicating that the activity was relatively hydrophilic. The activity in the HPLC peak lost most of its motion-stimulating ability after digestion with proteinase K. The motion stimulator could be a peptide analogous to the egg-associated peptides characterized in echinoderms which stimulate spermatozoa motion, respiration and chemotaxis.

Chemical Fractionation↗

PubMed1994

Comparison of the sensitivities of Salmonella typhimurium oxyR and katG mutants to killing by human neutrophils.

The respiratory burst of neutrophils is believed to kill bacteria by generating oxidative species, such as superoxide anion, hydrogen peroxide, and oxidized halogen species. The oxyR gene of Salmonella typhimurium controls a regulon induced by oxidative stress, such as exposure to hydrogen peroxide. Some researchers have suggested that oxyR may play a key role in bacterial survival following phagocytosis. We have tested this possibility by comparing the survival, following exposure to human neutrophils, of isogenic strains bearing different oxyR alleles. Neither inactivation of the oxyR gene nor constitutive overexpression of the oxyR-regulated proteins (oxyR1 allele) greatly alters bacterial resistance to neutrophils. The katG gene, encoding the oxyR-regulated enzyme hydroperoxidase I, was also without effect on survival following exposure to neutrophils. We conclude that the oxyR response does not play a significant role in the resistance of S. typhimurium to phagocytic killing in vitro.

Bacterial Proteins↗

PubMed1993

A new approach to measurement of redox-cycling activity in Escherichia coli.

Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species. Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells. E. coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration. We show that the use of this strain eliminates the requirement for cyanide in measurements of redox-cycling activity. The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption. The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E. coli strains. In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption.

Cyanides↗

PubMed1993

Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ) from IQ.

The mutagenic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following activation by ram seminal vesicle microsomes (RSVM, a source of prostaglandin H synthase, PHS) were studied in Salmonella typhimurium tester strains possessing elevated levels of acetyl-CoA: arylamine N-acetyltransferase (NAT). The metabolites formed by RSVM were extracted and fractionated by high pressure liquid chromatography (HPLC). One isolable product accounted for most of the direct-acting mutagenicity observed in the extracts. The metabolite was identified as 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ). Since nitro-IQ is a potent direct-acting mutagen, its role in IQ genotoxicity warrants further study.

Animals↗

PubMed1993

Mutational specificity of 2-nitro-3,4-dimethylimidazo[4,5-f]quinoline in the lacI gene of Escherichia coli.

2-Nitro-3,4-dimethylimidazo[4,5-f]quinoline (NO2-MeIQ) is the nitro derivative of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), one of the food pyrolysis product mutagens and carcinogens. The mutational specificity of NO2-MeIQ was determined using the lacI system in an Escherichia coli strain, EE125, harbouring the plasmid pKM101. NO2-MeIQ acts as a direct-acting mutagen towards this strain, and induces a broad range of mutational alterations. At higher doses; G:C-->T:A transversions dominate the mutational spectrum. Complex mutations were also observed, and the involvement of the SOS system in the processing of the mutations induced by NO2-MeIQ is suggested.

Bacterial Proteins↗

PubMed1993

Superoxide dismutase and the resistance of Escherichia coli to phagocytic killing by human neutrophils.

Transformation of Escherichia coli K-12-derived strains with a plasmid carrying the genetic determinants for synthesis of lipopolysaccharide O antigen by Shigella dysenteriae allows the construction of phenotypically smooth derivatives. We show that such E. coli K-12 derivatives are highly resistant to killing by human serum. Isogenic wild-type and sodB mutant (Fe superoxide dismutase-deficient) strains were constructed. The results of experiments on phagocytic killing of these strains by human neutrophils are reported. We observed no difference between the sensitivities of wild-type and sodB mutant strains to phagocytic killing, in contrast to the results reported by other researchers who used species other than E. coli or strains other than K-12.

Adult↗

PubMed1992

Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines.

Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl. Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized. Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant. However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase. The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities. In this paper we report the construction and use of new tester strains of S. typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme. These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines.

Arylamine N-Acetyltransferase↗

PubMed1992

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.

Arylamine N-Acetyltransferase↗

PubMed1990

Hydroperoxidase I catalyzes peroxidative activation of 3,3'-dichlorobenzidine to a mutagen in Salmonella typhimurium.

Dichlorobenzidine can be peroxidatively activated in Salmonella typhimurium Ames tester strains. Mutagenicity is observed when an S. typhimurium strain which is sensitive to frame-shift mutagens is incubated with dichlorobenzidine and hydrogen peroxide. In this paper, we show that the bacterial enzyme, hydroperoxidase I, is responsible for much of this activation. We constructed isogenic tester strains which lack hydroperoxidase I or II, due to Tn10 insertions in the structural genes encoding these proteins. Hydrogen peroxide-dependent mutagenicity of dichlorobenzidine was measured in each strain. A tester strain lacking hydroperoxidase I activity was much less sensitive than was the parent strain. When hydroperoxidase I activity was restored in this strain, via a plasmid-borne copy of the gene encoding the Escherichia coli protein, sensitivity to peroxide-dependent dichlorobenzidine mutagenicity was enhanced.

3,3'-Dichlorobenzidine↗

PubMed1990

Inhibition of benzo[a]pyrene dihydrodiol epoxide mutagenicity by synthetic analogues of ellagic acid.

Dibenzo[b, d]pyran-6-one, hydroxylated and methoxylated derivatives of this ring system, and some other analogues of the natural product ellagic acid have been synthesized and examined as inhibitors of benzo[a]pyrene dihydrodiol epoxide (BPDE) mutagenicity in Salmonella typhimurium strain TA100. Some of these new compounds have inhibitory effectiveness comparable to the natural product. On the basis of our results, we suggest qualitative rules for predicting inhibitory activity of ellagic acid analogues.

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

PubMed1990

Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli.

Escherichia coli responds to superoxide-generating agents by inducing approximately 40 proteins. We have identified a genetic locus, soxR (superoxide response), that positively regulates 9 of these proteins during superoxide stress. Induction under soxR control is at the transcriptional level, as shown with lac fusions to five paraquat-inducible promoters. Members of the soxR regulon include at least three proteins with demonstrable antioxidant roles: Mn-containing superoxide dismutase (which destroys superoxide radicals), endonuclease IV (which repairs radical-induced damages in DNA), and glucose-6-phosphate dehydrogenase (which produces NADPH). Induction of the soxR regulon also leads to diminished levels of the major outer membrane protein OmpF and alteration of the small-subunit ribosomal protein S6. These latter changes confer resistance to a variety of antibiotics. The soxR regulon may thus operate as an inducible defense against xenobiotics in general.

Anti-Bacterial Agents↗

PubMed1989

Dichlorobenzidine-DNA binding catalyzed by peroxidative activation in Salmonella typhimurium.

Peroxidative oxidation of dichlorobenzidine in vitro results in covalent binding to exogenous DNA. In a modified Ames assay, mutagenicity is observed in S. typhimurium strain TA98 following the incubation of dichlorobenzidine, bacteria, and hydrogen peroxide. In this paper, we demonstrate that [14C]dichlorobenzidine becomes covalently bound to S. typhimurium macromolecules, including DNA, when exogenous hydrogen peroxide is supplied. We compared the levels of binding in a pair of otherwise isogenic strains with wild-type (oxyR+) versus constitutive (oxyR1) expression of the hydrogen peroxide stress-induced regulon. Binding was approximately twofold higher in TA4124 (oxyR1) than in TA4123 (oxyR+). Bacterial hydroperoxidases may catalyze the activation of dichlorobenzidine to mutagenic and DNA binding species in this system.

3,3'-Dichlorobenzidine↗