Biomedical subjects

D M DeMarini

Publications and source records attributed to D M DeMarini.

At least 19 recordsLinked to original sources

PubMed2000

Mutation spectra of the drinking water mutagen 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) in Salmonella TA100 and TA104: comparison to MX.

The chlorinated drinking water mutagen 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) occurs at concentrations similar to or greater than that of the related furanone 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). MCF and MX differ structurally only by replacement of a 3-methyl in MCF with a 3-dichloromethyl in MX; yet, MCF is significantly less mutagenic than MX and produces different adducts when reacted with nucleosides or DNA. To explore further the effects that these structural differences might have on the biological activity of MCF and MX, we determined the mutation spectra of MCF in Salmonella strains TA100 and TA104 and of MX in strain TA104; the spectrum of MX in TA100 had been determined previously. In TA100, which presents only GC targets for mutagenesis, MCF induced primarily (75%) GC --> TA transversions, with most of the remaining revertants (20%) being GC --> AT transitions. This spectrum was not significantly different from that of MX in TA100 (P = 0.07). In TA104, which presents both GC and AT targets, MCF induced a lower percentage (57%) of GC --> TA transversions, with most of the remaining revertants (33%) being AT --> TA transversions. In contrast, MX induced almost only (98%) GC --> TA transversions in TA104, with the remaining revertants (2%) being AT --> TA transversions. Thus, almost all (98%) of the MX mutations were targeted at GC sites in TA104, whereas only 63% of the MCF mutations were so targeted. These results are consistent with the published findings that MX: (1) forms an adduct on guanosine when reacted with guanosine, (2) induces apurinic sites in DNA, and (3) forms a minor adduct on adenosine when reacted with adenosine or DNA. The results are also consistent with evidence that MCF forms adenosine adducts when reacted with adenosine. Our results show that the replacement of the 4-methyl in MCF with a 4-dichloromethyl to form MX not only increases dramatically the mutagenic potency but also shifts significantly the mutagenic specificity from almost equal targeting of GC and AT sites by MCF to almost exclusive targeting of GC sites by MX. Environ. Mol. Mutagen. 35:106-113, 2000 Published 2000 Wiley-Liss, Inc.

Alleles↗

PubMed1999

Induction of sister chromatid exchanges in human peripheral blood lymphocytes by bromoform: investigation of the role of GSTT1-1 polymorphism.

Brominated trihalomethanes (THMs) are disinfection by-products present frequently in chlorinated drinking water. Brominated THMs are mutagenic in a variety of systems and are carcinogenic in rodents. The metabolism of brominated THMs is thought to involve a GSH conjugation reaction leading either to formaldehyde or DNA-reactive intermediates via glutathione S-transferase-theta (GSTT1-1), which is polymorphic in humans. In the present study, we have determined the genotoxicity of one of the brominated THMs, bromoform (BF), by measuring its ability to induce sister chromatid exchanges (SCEs) in whole-blood (WB) cultures of human peripheral blood lymphocytes from GSTT1-1+ and GSTT1-1- donors. The results showed no differences in SCEs per cell by BF between GSTT1-1+ and GSTT1-1- individuals when the cells were exposed to 5 x 10(-3) M BF at the beginning of cell culturing (10.8+/-0.85 vs. 10.57+/-0.47, respectively), at the 16th (9.66+/-0.91 vs. 9.57+/-0.07), or the 24th h (8.21+/-0.61 vs. 8.29+/-0.24) of cell growth. Although GSTT1-1 is expressed in the erythrocytes, the lack of expression of the GSTT1-1 gene in the target cells (lymphocytes) may account for this observation.

Adult↗

PubMed1999

Mutation spectra in Salmonella TA98, TA100, and TA104 of two phenylbenzotriazole mutagens (PBTA-1 and PBTA-2) detected in the Nishitakase River in Kyoto, Japan.

Previous studies have identified two potent aromatic amine mutagens in the Nishitakase River, a tributary of the Yodo River, which serves as the main drinking water supply for the Osaka area in Japan. The two potent mutagens are 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). PBTA-1 and PBTA-2 are presumed to be formed from azo dyes discharged in a reduced form from dye factories to sewage treatment plants where they become chlorinated and are then discharged into the river. PBTA-1 and PBTA-2 account for 21% and 17% of the mutagenic activity of the Nishitakase River, respectively. Here we determined the mutation spectra induced by these two mutagens in TA98, TA100, and TA104 at 30-35, 8-10, and 2x, respectively, above the background. In TA98, the PBTA compounds produced identical mutation spectra, with 100% of the revertants containing the hotspot 2-base deletion of CG within the (CG)(4) sequence. In TA100, 73% of the revertants were GC-->TA transversions, with most of the remaining being GC-->AT transitions; the spectra produced by the two compounds in TA100 were not significantly different (p=0.8). In TA104, as in TA100, the majority (83%-87%) of the revertants were GC-->TA transversions, with most of the remaining revertants (11%-13%) being AT-->TA transversions. Thus, 83%-87% of the mutations induced by the PBTA compounds in TA104 were at G/C sites. The mutation spectra produced by the two compounds in TA104 were not significantly different (p0.08). PBTA-1 and PBTA-2 are structurally similar and have similar mutagenic potencies and mutation spectra in the respective strains. The mutation spectra produced by the PBTA compounds (100% hotspot deletion in TA98 and primarily GC-->TA transversions in TA100 and TA104) are similar to those produced by other potent aromatic amines, which is the class of compounds from which the PBTA mutagens derive.

Base Pairing↗

PubMed1999

Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3, 4-diepoxybutane in mouse, rat and human lymphocytes following in vitro G0 exposures.

To understand better the species differences in carcinogenicity caused by 1,3-butadiene (BD), we exposed G0 lymphocytes (either splenic or peripheral blood) from rats, mice and humans to 3, 4-epoxy-1-butene (EB) (20 to 931 microM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 320 uM), two of the suspected active metabolites of BD. Short EB exposures induced little measurable cytogenetic damage in either rat, mouse, or human G0 lymphocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in G0 splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approximately equisensitive to the cytogenetic damaging effects of DEB, but the situation for the human subjects was more complex. The presence of the GSTT1-1 gene (expressed in the erythrocytes) reduced the relative sensitivity of the lymphocytes to the SCE-inducing effects of DEB. However, additional factors also appear to influence the genotoxic response of humans to DEB. This study is the first direct comparison of the genotoxicity of EB and DEB in the cells from all three species.

Animals↗

PubMed1999

Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells.

Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

Animals↗

PubMed1999

Cytosol is required for the modulation by dietary casein of the hepatic microsomal activation of aflatoxin B1 to mutagenic metabolites detectable in Salmonella.

We have shown previously that dietary protein (casein) levels can affect the ability of rat liver S9 to metabolize aflatoxin B1 (AFB) as well as other promutagens detectable in Salmonella strain TA98 [Mutat. Res. (1997), 360, 115-126 and 127-143]. The mutagenic potency of AFB was greatest when metabolized by the Aroclor 1254-induced hepatic S9 prepared from F344 male rats that consumed an isocaloric, semisynthetic diet for 6 weeks that contained an adequate (12%) level of methionine-supplemented casein as the sole protein source, compared with S9s from rats fed diets that contained nominally deficient (8%) or high (22%) levels of casein. Here we have extended this observation by performing (i) mutagenicity studies with microsomes, cytosols and reconstituted S9s (recombinations of microsomes and cytosols across dietary groups), and (ii) in vitro incubations followed by analysis of metabolites by fluorescence high-pressure liquid chromatography. Microsomes, but not cytosols, activated AFB; however, activation to the level observed with S9 occurred only when microsomes from the rats fed 12% casein were combined with cytosols from any dietary group. Consistent with the mutagenicity results, the greatest metabolism of the AFB parent compound and the highest level of the glutathione conjugate of the presumptively identified AFB-exo-8,9-epoxide (the ultimate mutagenic form of AFB) were produced by S9s from the rats fed the 12% casein diet. The levels of these metabolites and the mutagenicity of AFB changed in parallel with changes in dietary casein levels. In summary, cytosolic elements, which are not affected by dietary casein levels, interact with microsomal enzymes, which are modulated by dietary casein levels, to influence the ability of hepatic S9 to activate AFB to a mutagen.

Aflatoxin B1↗

PubMed1999

Induction of genetic damage in human lymphocytes and mutations in Salmonella by trihalomethanes: role of red blood cells and GSTT1-1 polymorphism.

The brominated trihalomethanes (THMs) are mutagenic and carcinogenic disinfection by-products frequently found in chlorinated drinking water. They can be activated to mutagens by the product of the glutathione S-transferase-Theta (GSTT1++-1) gene in Salmonella RSJ100, which has been transfected with this gene. To evaluate this phenomenon in humans, we have examined the genotoxicity of a brominated THM, bromoform (BF), using the Comet assay in human whole blood cultures exposed in vitro. No differences were found in the comet tail length between cultures from GSTT1-1(+) versus GSTT1-1(-) individuals (1.67 +/- 0.40 and 0.74 +/- 0.54 microm/mM, respectively, P = 0.28). The high variability was due to the relatively weak induction of comets by BF. Combining the data from both genotypic groups, the genotoxic potency of BF was 1.20 +/- 0.34 microm/mM (P = 0.003). GSTT1-1 is expressed in red blood cells but not in the target cells (lymphocytes), and expression within the target cell (as in Salmonella RSJ100) may be necessary for enhanced mutagenesis in GSTT1-1(+) relative to GSTT1-1(-) cultures. To examine this, we exposed Salmonella RSJ100 and a control strain not expressing the gene (TPT100) to the most mutagenic brominated THM detected in Salmonella, dibromochloromethane (DBCM), either in the presence or absence of S9 or red blood cells from GSTT1-1(+) or GSTT1-1(-) individuals. S9 did not activate DBCM in the non-expressing strain TPT100, and it did not affect the ability of the expressing strain RSJ100 to activate DBCM. As with S9, red cells from either genotypic group were unable to activate DBCM in TPT100. However, red cells (whole or lysed) from both genotypic groups completely repressed the ability of the expressing strain RSJ100 to activate DBCM to a mutagen. Such results suggest a model in which exposure to brominated THMs may pose an excess genotoxic risk in GSTT1-1(+) individuals to those organs and tissues that both express this gene and come into direct contact with the brominated THM, such as the colon. In contrast, those organs to which brominated THMs would be transported via the blood might be protected by erythrocytes. Such a proposal is reasonably consistent with the organ specificity of drinking water-associated cancer in humans, which shows slightly elevated risks for cancer of the colon and bladder but not of the liver.

Adult↗

PubMed1998

Multifocal accumulation of p53 protein in esophageal carcinoma: evidence for field cancerization.

A systematic characterization of the cancerization field of esophageal carcinoma based on p53 protein accumulation has not been reported previously. The present report presents such a study based on 50 specimens of esophageal squamous-cell carcinoma from northern China. To gain insight into the etiology of this disease among the 50 subjects, DNA was analyzed for a polymorphism of the aldehyde dehydrogenase-2 (ALDH2) gene, which has been associated with increased risk for esophageal cancer among alcohol-consuming patients in Japan. However, the frequency of this polymorphism among our subjects, 30% (15/50), was within published control frequencies for this allele, suggesting that this allele may not play a role in the etiology of esophageal cancer in this northern Chinese population. Immuno-histochemical staining showed that 66% of the tumors were p53+. Of 420 pieces near or adjacent to p53+ tumors, p53+ cells were present among 64% of basal-cell hyperplasia (BCH), 70% of dysplasia (DYS) and 88% of carcinoma in situ (CIS). Of 216 pieces near or adjacent to p53- tumors, p53+ frequencies were 25% of BCH, 25% of DYS and 0% of CIS. The proportion of BCH cells that were p53+ decreased at increasing distance from the tumor (p = 0.006). The sporadic distribution of p53+ cells and the distribution and frequency of p53+ precursor lesions support the view that accumulation of p53 protein is multifocal and occurs in precursor lesions in early stages of esophageal carcinogenesis.

Adult↗

PubMed1998

Dietary interventions of human carcinogenesis.

The diet is a complex mixture that is associated with approximately 30% of human cancer in the U.S. Extensive laboratory studies indicate that the diet is composed of many mutagens/carcinogens as well as antimutagens/anticarcinogens. Overwhelming evidence from epidemiological studies indicates that a diverse diet that is high in fruits and vegetables and low in certain fats, along with moderate caloric intake and exercise, is most closely associated with reduced cancer risk. Dietary intervention studies using complex food items (fruits, vegetables, and fats) support these epidemiological observations; dietary interventions using single compounds (vitamins, antioxidants, etc.) have generally not. Estimates suggest that appropriate dietary changes could reduce the percentage of deaths due to prostate, colorectal, pancreatic, and breast cancer by >/=50%.

Anticarcinogenic Agents↗

PubMed1998

Mutation spectra resulting from carcinogenic exposure: from model systems to cancer-related genes.

The events leading to cancer are complex and interactive. Alteration of cancer genes, such as the tumor suppressor gene p53, plays a central role in this process. Analysis of the frequency, type and site of mutations in important cancer-related genes may provide clues to the identification of etiological factors and sources of exposure. In this chapter we have selected a few examples of environmental human carcinogens and have attempted to use the knowledge of their mechanisms of mutagenesis, as derived from in vitro cell systems, as a key to understanding the complexity of p53 mutation spectra in tumors arising at the putative target organ. The analysis will focus on environmental exposure to UV radiation. The examples of tobacco smoke, dietary aflatoxin and vinyl chloride will be also briefly discussed.

Aflatoxin B1↗

PubMed1998

Mutation spectra of complex mixtures.

Mutation spectra of complex mixtures or simple binary mixtures have been determined thus far only in Salmonella. This review summarizes these studies, which have involved a variety of complex mixtures, chemical fractions of the mixtures, and single compounds that are representative of the primary chemical classes within the mixtures. For the particulate organics from urban air and municipal waste incinerator emissions, cigarette smoke condensate, and organic extracts of chlorinated drinking water, the mutation spectrum of the mixture reflected the dominance of one or a few chemical classes within the mixture. The mutation spectra of sunlight and cigarette smoke in Salmonella were similar to those found in the p53 gene of skin or lung tumors in people associated with exposure to these two mutagens. These data provide possible mechanistic links between the types of mutations induced by complex environmental mutagens in an experimental organism (Salmonella) and the types of mutations found in humans exposed to the same environmental mutagens. The primary class of mutation produced by many mutagens within a chemical class (and even among different classes) is frequently the same. These studies indicate that, to some extent, complex mixture-induced mutation spectra can be modeled by the predominant mutagenic chemical class in that mixture and even by a single model compound in that chemical class. Such information should be useful in the interpretation of mutation spectra in molecular epidemiological studies where the exposure is primarily to a complex mixture.

Air Pollutants↗

PubMed1998

Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds.

To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev. The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein. Only 0.4-3.9% were true rev. Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions. The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101). The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions. Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times. The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101. Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions. Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated. The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins.

Alcohol Oxidoreductases↗

PubMed1997

Pilot study of free and conjugated urinary mutagenicity during consumption of pan-fried meats: possible modulation by cruciferous vegetables, glutathione S-transferase-M1, and N-acetyltransferase-2.

Epidemiological and experimental evidence indicates that consumption of fried meats in conjunction with certain genotypes of phase I and II metabolism genes poses an elevated risk for colorectal cancer. Parallel to this, the consumption of cruciferous vegetables is associated with a reduced risk of colon cancer. Therefore, we designed a 6-week pilot feeding study to evaluate the effect of these variables on urinary mutagenicity, which is a biomarker associated with fried-meat consumption. Eight subjects were fed fried meats daily for six weeks; four ate cruciferous vegetables, and four ate non-cruciferous vegetables. Urinary mutagenicity was evaluated in the presence of S9 in strain YG1024 of Salmonella, which is a frameshift strain that overproduces acetyltransferase. C18/methanol extracts of 24-h urines collected once each week were tested unhydrolyzed (free mutagenicity) and hydrolyzed (total mutagenicity); the difference between the two was the conjugated mutagenicity. Although not significant, the levels of conjugated urinary mutagenicity doubled among crucifera consumers and decreased to 30% of the initial levels among non-crucifera consumers, suggesting the possibility that crucifera may enhance the level of conjugated urinary mutagenicity resulting from consumption of fried meats. Such an effect would be consistent with the documented ability of cruciferous vegetables to induce phase II enzymes. The NAT2 rapid phenotype was significantly associated with approximately 2-fold increases in conjugated (p = 0.05) and total (p = 0.004) urinary mutagenicity relative to NAT2 slow subjects, consistent with the elevated risk confirmed by the NAT2 rapid phenotype for colorectal cancer among meat consumers. An approximately 2-fold increase in urinary mutagenicity among the GSTM1- subjects relative to the GSTM1+ subjects approached significance for free (p = 0.18) and total (p = 0.13) urinary mutagenicity. This is the first report on (a) the mutagenicity of hydrolyzed urine, which was consistently more mutagenic than unhydrolyzed urine; (b) the potential enhancement of conjugated urinary mutagenicity by crucifera; and (c) the association of the rapid NAT2 and possibly the GSTM1- phenotype with elevated levels of fried meat-associated urinary mutagenicity.

Aged↗

PubMed1997

Urinary mutagenicity as a biomarker in workers exposed to benzidine: correlation with urinary metabolites and urothelial DNA adducts.

Urinary mutagenicity has been used in occupational and epidemiological studies for over two decades as a cost-effective, general biomarker of exposure to genotoxic agents. However, few studies have compared urinary mutagenicity to additional biomarkers determined among low- and high-exposed groups. To address this issue, we evaluated the relationship between urinary mutagenicity and other types of biomarkers in a cross-sectional study involving 15 workers exposed to the urinary bladder carcinogen benzidine (BZ, high exposure), 15 workers exposed to BZ-dyes (low exposure), and 13 unexposed controls in Ahmedabad, India. Urinary organics were extracted by C18/methanol and evaluated for mutagenicity in the presence of S9 in the Salmonella strain YG1024, which is a frameshift strain that overproduces acetyltransferase. The results were compared to biomarker data reported recently from the same urine samples (Rothman et al., Proc. Natl Acad. Sci. USA, 93, 5084-5089, 1996) that included a metabolite biomarker (the sum of the urinary levels of BZ + N-acetylbenzidine + N,N'-diacetylbenzidine) and a DNA adduct biomarker [a presumptive N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine (C8dG-ABZ) DNA adduct in exfoliated urothelial cells]. The mean +/- SE urinary mutagenicity (revertants/micromol of creatinine) of the low-exposure (BZ-dye) workers was 8.2 +/- 2.4, which was significantly different from the mean of the controls (2.8 +/- 0.7, P = 0.04) as was that of the mean of the high-exposure (BZ) workers (123.2 +/- 26.1, P < 0.0001). Urinary mutagenicity showed strong, positive correlations with urinary metabolites (r = 0.88, P < 0.0001) and the level of the presumptive C8dG-ABZ urothelial DNA adduct (r = 0.59, P = 0.0006). A strong association was found between tobacco use (bidi smoking) and urinary mutagenicity among the controls (r = 0.68, P = 0.01) but not among the exposed workers (r = 0.18, P = 0.11). This study confirms the ability of a biomarker such as urinary mutagenicity to detect low-dose exposures, identify additional genotoxic exposures among the controls, and correlate strongly with urinary metabolites and DNA adducts in the target tissue (urinary bladder epithelia) in humans.

Benzidines↗

PubMed1997

Glutathione S-transferase-mediated induction of GC-->AT transitions by halomethanes in Salmonella.

Halomethanes are among the most common mutagenic and carcinogenic disinfection by-products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH2Cl2) and bromodichloromethane (BrCHCl2) can be mediated by a theta-class glutathione S-transferase (GSTT1-1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base-substitution strain TA1535 (hisG46, rfa, delta uvrB), into which has been cloned the GSTT1-1 gene from rat. In the present report, we have extended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr3) and chlorodibromomethane (CICHBr2), are also mediated by GSTT1-1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr3 approximately CICHBr2 > BrCHCl2 approximately CH2Cl2. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by performing colony probe hybridizations of approximately 100 revertants induced by each compound. The majority (96-100%) of the mutations were GC-->AT transitions, and 87-100% of these were at the second position of the CCC/GGG target. In contrast, only 15% of mutants induced by CH2Cl2 were GC-->AT transitions in the absence of the GSTT1-1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1-1 to mediate the mutagenicity of these di- and trihalomethanes and the induction of almost exclusively GC-->AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST-mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH-DNA adducts.

DNA↗

PubMed1996

Effect of dietary casein levels on activation of promutagens in the spiral Salmonella mutagenicity assay. II. Studies with induced rat liver S9.

In the previous study (Mutation Res., this issue), we showed that increased levels of dietary casein as the sole protein source for male F344 rats decreased the ability of the uninduced liver S9s to activate 2-aminoanthracene (2AN) to a mutagen in strain TA98 using the spiral Salmonella mutagenicity assay. No effects of dietary casein levels were noted for the ability of uninduced liver S9s to activate the promutagens aflatoxin B1 (AFB) and benzo[a]pyrene (BAP). In the present study, we have extended this study to include liver S9s induced with either Aroclor 1254, phenobarbital or 3-methylcholanthrene (3MC). S9s were derived from individual male F344 rats fed for 6 weeks on semisynthetic diets containing 8%, 12% or 22% methionine-supplemented casein as the sole source of protein (diets were made isocaloric by adjusting the corn starch content). Rats were housed in large, raised-bed cages by groups of three/diet/inducing agent. S9 activation mixtures were prepared at 5 mg of S9 protein/ml of S9 mix. Slopes from the linear portions of the mutagenicity dose-response curves were analyzed by ANOVA comparisons. Assays used to elucidate the phase I activities of microsomal preparations were cytochrome P-450 content, cytochrome-c reductase activity, flavin-containing monooxygenase activity, 7-ethoxyresorufin O-deethylation (EROD) activity, N-demethylation of benzphetamine, and para-nitrophenol O-deethylation. Phase II activities were assayed by estimating glutathione (GSH) content and measuring the metabolism of 1-chloro-2,4-dinitrobenzene (CDNB) by glutathione S-transferase in cytosolic preparations. None of the phase I or phase II endpoints were significantly affected by dietary casein levels. In general, increasing levels of dietary casein resulted in increased body and liver wet weight and amount of S9 protein. Aroclor-induced S9s from rats fed the 22% or 12% casein diet were most effective at activating AFB, depending on the lot of Aroclor used for induction; these divergent results were replicated with two groups of rats for each lot of Aroclor. The observed differences between Aroclor lots are assumed to arise from variation in the mix of PCB isomers. The Aroclor-induced S9s did not exhibit any casein-related effects for the activation of BAP or 2AN. For 3MC-induced S9s, the 12% casein diets produced S9s with the highest ability to activate AFB and BAP when standardized for protein content. Phenobarbital-induced S9s did not demonstrate any dietary casein-related effects on the activation of the three model promutagens. These results illustrate the complex interaction between dietary levels of casein, enzyme inducing agent and promutagen.

Aflatoxin B1↗

PubMed1996

Effect of dietary casein levels on activation of promutagens in the spiral Salmonella mutagenicity assay. I. Studies with noninduced rat liver S9.

Xenobiotic metabolism can be influenced by various nutritional factors, including protein. In the present study, we have examined the effect of dietary protein (casein) levels on the ability of rat liver S9 to activate the promutagens aflatoxin B1 (AFB), 2-aminoanthracene (2AN) and benzo[a]pyrene (BAP) in strain TA98 using the spiral Salmonella mutagenicity assay. S9s were derived from individual male F344 rats fed for 6 weeks on semisynthetic diets containing 8%, 12% or 22% methionine-supplemented casein as the sole source of protein (diets were made isocaloric by adjusting the corn starch content). Rats were housed in large, raised-bed cages by groups of three per diet. S9 activation mixtures were prepared at 5 mg of S9 protein/ml of S9 mix. Slopes from the linear portions of the mutagenicity dose-response curves were analyzed by ANOVA comparisons. Assays used to elucidate the phase I activities of microsomal preparations were cytochrome P450 content, cytochrome-c reductase activity, flavin-containing monooxygenase activity, 7-ethoxyresorufin O-deethylation (EROD) activity, N-demethylation of benzphetamine and para-nitrophenol O-deethylation. Phase II activities in cytosolic preparations were assayed by estimation of glutathione (GSH) content and glutathione S-transferase activity through metabolism of 1-chloro-2,4-dinitrobenzene (CDNB). Increased levels of dietary casein increased liver wet weights and decreased the ability of the S9 to activate 2AN. Dietary casein levels did not influence the S9-mediated activation of BAP; and consistent but nonsignificant increases in activation of AFB were produced by S9 from animals fed the 22% casein diet. The phase I and phase II activities measured here were not altered significantly by dietary casein levels; thus, other, more specific enzymatic activities may account for the mutagenesis data. These results illustrate the complex interaction between dietary levels of casein and promutagen activation mechanisms, which prevents drawing broad generalizations regarding the influence of dietary casein levels on the capacity of hepatic S9s to activate promutagens.

Aflatoxin B1↗

PubMed1996

Mutation spectra of chemical fractions of a complex mixture: role of nitroarenes in the mutagenic specificity of municipal waste incinerator emissions.

Using an ion-exchange procedure coupled to a microsuspension Salmonella assay, we fractionated the dichloromethane-extractable particulate organics emitted by a municipal waste incinerator. Most (80-95%) of the mutagenic activity resided in the neutral/base fraction; however, the polar neutral fraction accounted for 12% of the direct-acting mutagenic activity. The mutagenic potencies of the whole extract and the various fractions were 4-15 times greater in the absence than in the presence of S9. Results with strains deficient in classical nitroreductase (TA98NR) and transacetylase (TA98/1,8-DNP6) indicated that a majority of the direct-acting mutagenicity was due to nitroarenes. This was confirmed by bioassay-directed subfractionation of the neutral/base faction by a cyanopropyl/HPLC method. The mutations in -3,000 revertants (approximately 400 each induced in TA98 by the whole extract, the neutral/base and polar neutral fractions from the ion-exchange column and 3 of the neural/base subfractions from the HPLC column; along with 200 revertants each induced by the model nitroarene 1-nitropyrene (1NP) in strains TA98, TA1538 and TA100) were analyzed by probe hybridization and PCR/DNA sequence analysis. The results indicated that nitroarenes such as 1NP that eluted in the neutral/base fraction accounted for at least 50% of the direct-acting mutagenicity and induced only a hotspot 2-base deletion in the sequence (CG)4 in TA98. In contrast, most of the complex frameshifts (a frameshift with a flanking base substitution) induced by the whole extract were induced by nitroarenes other than 1NP that were activated by transacetylation and that eluted in the polar neutral fraction. This study (1) identifies nitroarenes as an important contributor to the mutagenic activity of the emissions from municipal waste incinerators; (2) confirms our previous conclusion that the mutation spectrum of a complex mixture reflects the dominance of particular classes of chemical mutagens within the mixture; and (3) demonstrates the possibility of isolating certain chemical fractions of a complex mixture that induce certain classes of mutations produced by the whole, unfractionated mixture.

Air Pollutants↗