Mutagenic potential of sediments from the Grand Calumet River.
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Mutagenicity Tests↗
Biomedical subjects
A E Maccubbin
Publications and source records attributed to A E Maccubbin.
Altered benzo[a]pyrene metabolism in C3H/10T1/2 cells transformed by aflatoxin B1 or 3-methylcholanthrene.
C3H/10T1/2 clone 8 (10T1/2) cells possess Phase I and Phase II xenobiotic metabolizing enzymes associated with the metabolism of polycyclic aromatic hydrocarbons to activated or detoxified species. We compared the metabolism of benzo[a]pyrene (BaP) by these cells to an aflatoxin B1 (AFB1)-transformed line (7SA) and a 3-methylcholanthrene (3-MC)-transformed line (MCA) isolated from carcinogen-treated 10T1/2 cells. Relative to 10T1/2 cells, basal levels of cytochrome P450-mediated aryl hydrocarbon hydroxylase (AHH) were significantly depressed in 7SA cells by about 30%. The inducibility of AHH by BaP treatment was depressed by 30-70% in MCA and 7SA cells over a 36-hr time course. 10T1/2 and MCA cells accumulated similar intracellular amounts of 3-OH-BaP by 12 and 24 hr, respectively; in contrast the accumulation of 3-OH-BaP in 7SA cells was 70% lower. During 36 hr of BaP treatment, total BaP-DNA adduct levels formed in 7SA and MCA cells, determined by 32P-postlabeling analysis, were 90 and 83% lower, respectively, than those found in 10T1/2 cells. These differences in response to BaP treatment were not related to cellular differences in the uptake or efflux of BaP. Relative to 10T1/2 or MCA cells, 7SA cells were found to have at least a twofold increase in UDP-glucuronyltransferase activity, which correlated with the lower intracellular accumulation of 3-OH-BaP and enhanced formation of extracellular polar metabolites. MCA cells had an almost twofold increase in glutathione S-transferase activity relative to parental 10T1/2 cells but produced lower levels of extracellular polar metabolites. These results demonstrate an association between chemical transformation of 10T1/2 cells and altered xenobiotic metabolism. This system may provide an in vitro model for examining the molecular events responsible for the biochemically altered phenotype of the malignantly transformed cell.
Aflatoxin B1↗
32P-postlabeling detection of DNA adducts in fish from chemically contaminated waterways.
Fish were collected from sites in the chemically-contaminated Buffalo River, New York, and the Detroit River, Michigan. The sediments of these rivers have high levels of chemical contaminants, including polycyclic aromatic hydrocarbons (PAHs), and fish from these locations have high prevalences of liver cancer. To determine chemical-DNA interactions and a possible role for chemicals as a cause of the observed tumors, DNA was isolated from livers and was enzymatically digested to normal and adducted nucleotides. The DNA digests were enriched for hydrophobic, bulky adducts, either by preparative reverse phase high pressure liquid chromatography, or by selective nuclease P1 dephosphorylation of normal nucleotides. DNA-chemical adducts were then quantitated by 32P-postlabeling analysis. Regardless of the adduct enrichment procedure, the chromatograms derived from DNA of fish from polluted areas showed a diffuse, diagonal radioactive zone consisting, at least in part, of multiple overlapping discrete adduct spots. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky, hydrophobic, aromatic genotoxic compounds. Analysis of bile demonstrated recent exposure to multi-ringed aromatic compounds.
Animals↗
Acute toxicity and mutagenicity of the copper complex of pyruvaldehyde-bis (N-4-methylthiosemicarbazone), Cu-PTSM.
Cu-PTSM is a potential imaging agent for the heart and brain when labeled with either 64Cu or 62Cu. Unlabeled Cu-PTSM was evaluated for its acute toxicity and mutagenicity. Cu-PTSM had an i.v. LD50 of 26 mg kg-1 in the rat and 2 mg kg-1 in the rabbit. At necropsy, rats exhibited severely hemorrhagic lungs, histological findings of acute pulmonary congestion, hemorrhage and edema, and mild congestion in kidney, liver and brain. The rabbit displayed marked polymorphonuclear infiltration in alveoli, peribronchial and periarterial areas with marked macrophage hyperplasia, congestion and mild hemorrhage into alveolar spaces. No effects were found in kidney, liver, testes or brain. Administration of 2.16 micrograms kg-1 day-1 for 5 days per week for 2 weeks resulted in no changes in histopathology, hematology or clinical chemistry parameters. This daily dose is at least 300 times the diagnostic dose intended for use in man. Cu-PTSM was not mutagenic when tested in the absence of S9 supernatant, but elicited a weakly mutagenic response in the presence of S9. Since acute effects in the lung occur at doses approaching 300,000 times the diagnostic dose, it is highly unlikely that the clinical use of Cu-PTSM would result in any acute adverse effects.
Animals↗
32P-postlabeling analysis of binding of the cyclophosphamide metabolite, acrolein, to DNA.
The cyclophosphamide metabolite, acrolein, was reacted with 2'-deoxyguanosine-3'-monophosphate, and two adducts were detected by high performance liquid chromatography and 32P-postlabeling assay. These adducts were resistant to dephosphorylation by nuclease P1 and could be isolated and detected from calf thymus DNA that had been reacted in vitro with acrolein. A combination of HPLC purification and enzymatic digestion of normal nucleotides by nuclease P1 allowed for the detection of these adducts in hepatic DNA from mice treated with cyclophosphamide. The level of the two adducts in the hepatic DNA, as determined by 32P-postlabeling, was one adduct per 2.7-4.1 x 10(7) normal nucleotides.
Acrolein↗
Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine.
The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32p from [gamma 32P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32p-postlabeling assay.
Alkylating Agents↗
Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene.
C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.
Aflatoxin B1↗
Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.
Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.
Aflatoxin B1↗
The genetics of aflatoxin B1 metabolism. Association of the induction of aflatoxin B1-4-hydroxylase with the transcriptional activation of cytochrome P3-450 gene.
The association between murine cytochrome P3-450 and hepatic aflatoxin B1-4-hydroxylase, a cytochrome P-450-dependent enzyme which converts aflatoxin B1 (AFB1) to aflatoxin M1 (AFM1), was examined by (a) purification of the cytochrome P-450 which preferentially metabolizes AFB1 to AFM1; (b) isolation of the specific cDNA clone; and (c) correlating induction of transcriptional activation of the specific message with the enzyme activity in the hepatic microsomes. Isolation of cytochromes P-450 from C57BL/6 mice, an Ah-responsive strain, pretreated with a 150 mg/kg dose of beta-naphthoflavone resulted in the partial purification of the cytochrome P-450 with preference for the metabolism of AFB1 to AFM1. Antibodies raised against this cytochrome P-450 were used to enrich hepatic mRNA for cDNA cloning. A cDNA library screened with a rat cytochrome P-450c gene probe yielded only two types of cDNA clones that contained inserts corresponding to cytochrome P1-450 and cytochrome P3-450. Specific restriction fragments of near full-length P1-450 cDNA and full-length P3-450 cDNA, hybridizing only with their respective messages, were isolated and used to assess transcriptional activation of these messages in liver and extrahepatic tissues from C57BL/6 mice treated with 3-methylcholanthrene, beta-naphthoflavone, indolylacetonitrile, and Aroclor-1254. Dose-dependent induction of the two messenger RNAs, when compared with the induction of specific enzyme activities, demonstrated the association of cytochrome P1-450 with aryl hydrocarbon hydroxylase activity and the association of cytochrome P3-450 with AFB1-4-hydroxylase activity. This supports our earlier hypothesis that AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase, although regulated by the Ah locus, are the products of two separate genes (Gurtoo, H.L., Dahms, R.P., Kanter, P., and Vaught, J.B. (1978) J. Biol. Chem. 253, 3952-3961).
Aflatoxin B1↗
Aflatoxin B1 induced hepatic neoplasia in Great Lakes coho salmon.
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Aflatoxin B1↗
The toxicity and mutagenicity of S-3-iodo-N-(1-ethyl-2-pyrrolidinyl)methyl-2-hydroxy-6-methoxybenzamide (IBZM), a new CNS D-2 dopamine receptor imaging agent.
IBZM is one of several benzamide derivatives showing a high affinity for the CNS D-2 dopamine receptor. Carrier-free [123I]IBZM is potentially useful as a nuclear medicine imaging agent for investigating CNS D-2 dopamine receptor in humans. This study describes the acute toxicity of IBZM in the rat and rabbit, its subchronic toxicity in the rabbit and its mutagenicity measured by the Ames test. IBZM had a 24 hour LD50 of 400 mg/kg in the rat and 50 mg/kg in the rabbit when administered i.v. Deaths occurred within minutes of dosing. Some necrosis was evident at the injection site in IBZM treated animals which was not found in the controls. No gross or histological differences between experimental animals and controls were evident in surviving animals when necropsied 14 days after dosing. Repeated exposure of rabbits to IBZM at a total cumulative dose of 100,000 times the expected clinical dose revealed no consistent changes in hematology, blood chemistry, blood enzymes or tissue pathology. IBZM was not mutagenic in the modified Ames assay with or without metabolic activation in the TA98 and TA100 tester strains. It is therefore unlikely that acute adverse effects will be associated with the diagnostic use of this drug in man.
Animals↗
Evidence for polynuclear aromatic hydrocarbons in the diet of bottom-feeding fish.
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Animals↗
A reliable, efficient, microinjection apparatus and methodology for the in vivo exposure of rainbow trout and salmon embryos to chemical carcinogens.
A modular apparatus and technique for the injection of salmonid fish embryos with chemical carcinogens are described. A key feature of the methodology is the relative ease of routine through-the-eggshell injection, into the yolk sac of living salmonid fish embryos, inside the "eyed-stage" egg. The procedure is sufficiently rapid that 2 persons working as a team can give injections to 200 embryos per hour. The injection per se induces low mortality, i.e., optimal net survival rates (controls given an injection of dimethyl sulfoxide vs. those not given an injection) in the range of 70-90%. Because only small amounts of chemical are handled in relatively dilute form, the exposure method poses low risks to both the experimentalist and the environment. Preliminary results in a test of 4 carcinogens that differed widely in their structures and requirements for metabolic activation indicated that hepatocellular neoplasms were induced in rainbow trout (Salmo gairdneri) in response to 100 ng aflatoxin B1 (CAS: 1162-65-8)/egg, 1 microgram N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7)/egg, and 10 micrograms benzo[a]pyrene (CAS: 50-32-8)/egg. Nine months after exposure, liver neoplasms were observed in 25, 21, and 9%, respectively, of the rainbow trout, but no neoplasms were observed in rainbow trout exposed to 100 micrograms dimethylnitrosamine (CAS: 62-75-9)/egg. Liver neoplasms were also induced in 17% of coho salmon (Oncorhynchus kisutch) given an injection as embryos of 90 ng aflatoxin B1/egg or 5 micrograms N-methyl-N'-nitro-N-nitrosoguanidine/egg.
Aflatoxin B1↗
Preparation, characterization, and microbial degradation of specifically radiolabeled [C]lignocelluloses from marine and freshwater macrophytes.
Specifically radiolabeled [C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [C]phenylalanine, [C]tyrosine, and [C]cinnamic acid as precursors. Specifically radiolabeled [C-polysaccharide]lignocelluloses were prepared by using [C]glucose as precursor. The rates of microbial degradation varied among [C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [C]phenylalanine and [C]tyrosine were found associated with protein, although very little (3%) radioactivity from [C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to CO(2); during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.
Journal Article↗
Anaerobic biodegradation of the lignin and polysaccharide components of lignocellulose and synthetic lignin by sediment microflora.
Specifically radiolabeled [C-lignin]lignocelluloses and [C-polysaccharide]lignocelluloses were prepared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush, and a hardwood. These [C]lignocellulose preparations and synthetic [C]lignin were incubated anaerobically with anoxic sediments collected from a salt marsh, a freshwater marsh, and a mangrove swamp. During long-term incubations lasting up to 300 days, the lignin and polysaccharide components of the lignocelluloses were slowly degraded anaerobically to CO(2) and CH(4). Lignocelluloses derived from herbaceous plants were degraded more rapidly than lignocellulose derived from the hardwood. After 294 days, 16.9% of the lignin component and 30.0% of the polysaccharide component of lignocellulose derived from the grass used (Spartina alterniflora) were degraded to gaseous end products. In contrast, after 246 days, only 1.5% of the lignin component and 4.1% of the polysaccharide component of lignocellulose derived from the hardwood used (Rhizophora mangle) were degraded to gaseous end products. Synthetic [C]lignin was degraded anaerobically faster than the lignin component of the hardwood lignocellulose; after 276 days, 3.7% of the synthetic lignin was degraded to gaseous end products. Contrary to previous reports, these results demonstrate that lignin and lignified plant tissues are biodegradable in the absence of oxygen. Although lignocelluloses are recalcitrant to anaerobic biodegradation, rates of degradation measured in aquatic sediments are significant and have important implications for the biospheric cycling of carbon from these abundant biopolymers.
Journal Article↗
Relative contributions of bacteria and fungi to rates of degradation of lignocellulosic detritus in salt-marsh sediments.
Specifically radiolabeled [C-lignin]lignocellulose and [C-polysaccharide]lignocellulose from the salt-marsh cordgrass Spartina alterniflora were incubated with an intact salt-marsh sediment microbial assemblage, with a mixed (size-fractionated) bacterial assemblage, and with each of three marine fungi, Buergenerula spartinae, Phaeosphaeria typharum, and Leptosphaeria obiones, isolated from decaying S. alterniflora. The bacterial assemblage alone mineralized the lignin and polysaccharide components of S. alterniflora lignocellulose at approximately the same rate as did intact salt-marsh sediment inocula. The polysaccharide component was mineralized twice as fast as the lignin component; after 23 days of incubation, ca. 10% of the lignin component and 20% of the polysaccharide component of S. alterniflora lignocellulose were mineralized. Relative to the total sediment and bacterial inocula, the three species of fungi mediated only very slow mineralization of the lignin and polysaccharide components of S. alterniflora lignocellulose. Experiments with uniformly C-labeled S. alterniflora material indicated that the three fungi and the bacterial assemblage were capable of degrading the non-lignocellulosic fraction of S. alterniflora material, but only the bacterial assemblage significantly degraded the lignocellulosic fraction. Our results suggest that bacteria are the predominant degraders of lignocellulosic detritus in salt-marsh sediments.
Journal Article↗
Mineralization of detrital lignocelluloses by salt marsh sediment microflora.
Specifically radiolabeled C-(cellulose)-lignocellulose and C-(lignin)-lignocellulose were isolated from labeled cuttings of Spartina alterniflora (cordgrass) and Pinus elliottii (slash pine). These were used to estimate the rates of mineralization to CO(2) of lignocelluloses of estuarine and terrestrial origin in salt marsh estuarine sediments. The lignin moiety of pine lignocellulose was mineralized 10 to 14 times more slowly than that of Spartina lignocellulose, depending on the source of inoculum. Average values for percent mineralization after 835 h of incubation were 1.4 and 13.9%, respectively. For Spartina lignocellulose, mineralization of the cellulose moiety was three times faster than that of the lignin moiety. Average values for percent mineralization after 720 h of incubation were 32.1 and 10.6%, respectively. Lignocellulose and lignin contents of live pine and Spartina plants were analyzed and found to be 60.7 and 20.9%, respectively, for pine and 75.6 and 15.1%, respectively, for Spartina.
Journal Article↗