Acute hyperplasia and peroxisome proliferation induced by methylclofenapate: a species comparison and implications for liver carcinogenesis.
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Animals↗
Biomedical subjects
J A Styles
Publications and source records attributed to J A Styles.
A species comparison of acute hyperplasia induced by the peroxisome proliferator methylclofenapate: involvement of the binucleated hepatocyte.
The acute hyperplastic response induced by methylclofenapate (MCP) was studied in several rodent species with different responsiveness to the hypertrophic and hyperplastic effects caused by this chemical. The species, in descending order of responsiveness, were: mouse, rat, hamster and guinea-pig, the latter species being non-responsive. Animals were dosed at daily intervals with MCP (25, 12 or 5 mg/kg by gavage) and killed at intervals from 12 h to 240 h. The parameters of ploidy, nuclearity and DNA synthesis were examined in isolated hepatocytes. The hyperplastic response elicited by MCP in rodent livers as detected by the occurrence of S-phase cells, was almost exclusively confined to the 2 x 2N (binucleated) hepatocyte population. At the same time the proportion of 2 x 2N cells was reduced in a time and dose-dependent manner, while the fraction of 4N cells increased. These observations indicate that the 2 x 2N cells responding to MCP undergo S-phase followed by amitotic cytokinesis to form 4N cells. The response in rats, mice and hamsters was quantitatively different but qualitatively similar, while the guinea-pig was non-responsive. In a given responsive species the areas under the curves are similar for different doses, indicating that the size of the responsive population is limited. The data also indicate that although the response in the mouse was greater than in the rat, because the total number of responsive 2 x 2N cells is larger, the percentage of responsive 2 x 2N cells is higher in the rat than in the mouse. The ploidy analysis reveals that there is no detectable change in the ratio of (4N + 2 x 2N):2N hepatocytes, but only 1-2% change would be expected, despite the number of 4N cells produced, due to the increase in total cell number.
Animals↗
A cytological comparison between regeneration, hyperplasia and early neoplasia in the rat liver.
Regeneration, hyperplasia and neoplasia are three different responses to injury in the rat liver. These phenomena were induced in rat liver and the parameters of ploidy, nuclearity and DNA synthesis were examined. Analysis of hepatocytes from animals undergoing liver regeneration following two-thirds partial hepatectomy revealed that there is an increase in the cycling of diploid hepatocytes and a large increase in the frequency of binucleated tetraploid cells undergoing DNA synthesis and amitotic cytokinesis to mononucleated tetraploid cells. This results in an overall increase in the ratio of tetraploid:diploid cells but no change in the proportion of binucleated cells. The liver appears, temporarily, to undergo an increased rate of maturation. In both hyperplasia inducted by oral administration of 25 mg/kg methylclofenapate or diethylhexylphthalate (1 g/kg for 4 weeks) and neoplasia induced by the hepatocarcinogens 3'-methyl-4-dimethylaminoazobenzene (3'M), 6-p-dimethylaminophenylazobenzthiazole (6BT), 5-phenylazoindazole (5I), diethylnitrosamine (DEN) and thioacetamide (TA) the binucleated cell is sensitive to the action of the chemicals, although its response is different. Both types of carcinogen induce a reduction in the frequency of binucleated cells but the mononucleated diploid cells produced by cytokinesis without a preceding S phase as a result of the action of genotoxic carcinogens appear to be incapable of polyploidization and give rise to a liver with a permanently depressed tetraploid:diploid hepatocyte ratio. The nongenotoxic carcinogens methylclofenapate and DEHP cause an initial hyperplastic response due to the rapid conversion of binucleated cells to mononucleated tetraploids by amitotic cytokinesis following S phase. Over a longer period of exposure there is an increase in the tetraploid:diploid ratio due to the continued conversion of newly formed binucleates to tetraploid mononucleates.
Animals↗
The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens.
The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.
Animals↗
Activity of bromochlorodifluoromethane (BCF) in three mutation tests.
The halocarbon BCF was tested in 3 assays to assess its mutagenicity and clastogenicity. It produced a positive response in Salmonella typhimurium strain TA1535 but was negative in TA1537, TA1538, TA98 and TA100. In an L5178Y mouse lymphoma microwell assay (TK locus), BCF was negative. BCF was administered at 5000 and 50 000 ppm in air for 6 h to groups of C57B1/6J mice of both sexes. Animals were killed at 24, 48 and 72 h after cessation of exposure and the incidence of bone marrow micronuclei per 1000 PCEs determined. There was no significant difference in the incidences of micronuclei between untreated animals and those exposed to either concentration of BCF at any of the sampling times. These results suggest that BCF is mutagenic in vitro in only one strain of Salmonella; in mammalian cells the compound induced no gene mutation in vitro nor clastogenic activity in vivo at doses that also produced clear evidence of toxicity.
Animals↗
Genotoxicity and carcinogenicity of fluorocarbons: assessment by short-term in vitro tests and chronic exposure in rats.
Two short-term in vitro tests for mutagenicity (Salmonella reverse mutation and BHK21 cell transformation) were conducted on a series of fluorocarbons. Some of these materials (FC22, FC31, FC142b, FC143, and FC143a) were found to be positive in one or both of the tests and could therefore be considered as being potentially carcinogenic to animals. Such activity was not anticipated for what were previously considered inert materials and in consequence several examples of these fluorocarbons, which represented different combinations of short-term test results, were tested for carcinogenicity in limited in vivo bioassays. In these studies, rats were dosed for 1 year by gavage 5 days a week with either FC22, FC31, FC133a, FC134a, or FC143a dissolved in a corn-oil at a single dosage of 300 mg/kg body weight. The animals were then observed until week 125 with detailed necropsy at termination. The study revealed that FC31 was a potent carcinogen (to the rat stomach), a result which reflected the short-term test predictions, but FC133a, which gave a negative response in both the in vitro assays, induced a high incidence of reproductive tract tumors. The weak bacterial mutagens FC22 and FC143a did not induce tumors in this study, and the nonmutagenic FC134a was without overt carcinogenic activity. It is concluded that, while recognizing the limitations of the in vivo component of this study, the short-term tests were only partially successful in identifying potential carcinogens for this series of chemicals. Fluorocarbon 31 was a potent carcinogen which was first identified by bacterial mutation and cell transformation, whereas the equally potent carcinogen FC133a was not so identified. The lack of genotoxic activity with this particular compound leads us to believe that the carcinogenic activity may be due to mechanisms other than those which involve direct DNA interactions.
Animals↗
Cytogenetic effects of benzene: dosimetric studies on rats exposed to benzene vapour.
Rats were exposed to benzene vapour at nominal concentrations in air of 1, 10, 100 and 1000 ppm acutely for 6 h. Bone marrow cells from each animal were examined for chromosomal abnormalities 24 h after the end of the exposure period. This analysis was carried out on 250 metaphases per animal where possible and showed a significant increase in the percentage of cells with chromosomal abnormalities, excluding gaps, in the groups of animals exposed to 100 and 1000 ppm benzene. In the 10-ppm and 1-ppm exposure groups there were elevated levels of cells with abnormalities which showed evidence of being dose-related, although they were not statistically significant.
Animals↗
Effects of hydroxyurea consistent with the inhibition of repair synthesis in ultraviolet irradiated HeLa cells.
Many studies on DNA repair using established in vitro cell cultures employ conditions of low serum, nutrient deprivation, and blockade with hydroxyurea (HU) to reduce the background levels of DNA replication. There are some reports in the literature which indicate that HU inhibits DNA repair. In the present study the effects of HU on strand breaks and repair synthesis in UV irradiated Hela cells were investigated using a combined strand break and UDS assay. In conditions of low serum and arginine deprivation, HU produced effects consistent with the inhibition of the repair synthesis step in excision repair. Although the conditions used in this study are severe the results suggest that HU may have qualitatively similar effects in other studies which employ its use to detect repair synthesis.
DNA↗
Resistance of mouse lymphoma L5178YAII cells to alkylation with methylmethane sulphonate resides in a late step of excision repair.
The mutant mouse lymphoma cell line (L5178YAII), resistant to X-rays, ultraviolet light and alkylating agents, was reinvestigated in an attempt to establish the nature of the mutation. These cells were compared with P388 mouse lymphoma cells, which exhibit normal sensitivity to these mutagens. A series of studies was conducted to compare DNA alkylation and strand breakage with cell survival after exposure of the two cell lines to methylmethane sulphonate. It was found that neither the degree of alkylation nor the removal of the common alkylation products was correlated with the different sensitivities observed in these cell lines. A correlation was established between cell killing and the production of long-lived strand breaks. P388 cells were found to accumulate twice as many long-lived strand breaks compared to L5178YAII cells, at equal levels of alkylation. This suggested that long-lived strand breaks were the major toxic lesions. Further experiments indicated that these long-lived strand breaks were produced by a process consistent with excision repair. Evidence is also presented that indicates that the mutation in L5178YAII cells that is responsible for their resistance may occur in ligase activity or its associated ADP-ribosyl transferase system.
Alkylation↗
Evaluation of some formaldehyde-release compounds and other biocides in the mouse micronucleus test.
A number of biocidal chemicals were tested for clastogenic activity in the micronucleus test using C57Bl/6J mice. The materials tested were: 5-chloro-2-methyl-4-isothiazolin-3-one (I), N-methyl-isothiazolone hydrochloride (II), Glokill 77 and Parmetol A23. Two of the biocides (Glokill and Parmetol) depend on the release of formaldehyde for their activity while the other two compounds are the active chemicals in the biocide Kathon. Hexamethylphosphoramide (HMPA) was tested as the positive control for the series and N,N-dinitrosopentamethylenetetramine (DNPT) as the negative control. HMPA produced significant dose-related increases in the incidence of micronuclei whereas DNPT, I, II, Glokill and Parmetol A23 were without effect.
Animals↗
Activity of vinyl chloride monomer in the mouse micronucleus assay.
C57Bl/6J mice of both sexes were exposed to 50 000 ppm vinyl chloride monomer (VCM) for 6 h. Animals were killed 24 and 48 h after cessation of exposure and examined for the presence of micronuclei in bone marrow cells. At 24 h the control incidences of micronuclei per 1000 polychromatic erythrocytes (PCEs) were 2.6 (male) and 1.2 (female), while in animals exposed to VCM the incidences were 24.6 (male) and 25.0 (female). At 48 h the control incidences were 2.2 (male) and 1.6 (female) and in the VCM exposed animals 7.2 (male) and 4.4 (female).
Animals↗
A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyclophosphamide or hexamethylphosphoramide (HMPA).
The response of 3 strains of mouse (C57Bl/6J, C3H/C57 hybrid and BALBC/CBA) to cyclophosphamide (75 mg/kg) and hexamethylphosphoramide (HMPA) (1.28 ml/kg) were compared in the micronucleus test. Each compound was administered by intraperitoneal injection on two consecutive days and samples of bone marrow and blood taken for examination at 48 and 72 h after the first injection. Both test chemicals produced a statistically significant increase (P 0.001) in the incidence of micronuclei in bone marrow cells in all strains at both sampling times but the response with HMPA in C57Bl/6J mice appears to occur earlier than in the other two strains. Significant increases in micronuclei were seen in circulating erythrocytes only at 48 h in C57Bl/6J mice with both test chemicals and in C3H/C57 mice only with cyclophosphamide.
Animals↗
Activity of 2,4,6-trinitrotoluene in an in vitro mammalian gene mutation assay.
In the present study both 2,4,6-trinitrotoluene (TNT) and pure 2,4-dinitrotoluene (2,4-DNT) gave positive responses in the P388 mouse lymphoma gene mutation assay in the absence of auxiliary metabolic activation. Both chemicals gave negative results when an activation system was included. Technical grade DNT (consisting of an 80:20 mixture of 2,4- and 2,6-DNT) and pure 2,6-DNT gave negative responses in the assay both in the presence and absence of auxiliary metabolism. These observations in mammalian cells support the bacterial mutagenesis data indicating the TNT is a potential rodent liver carcinogen and suggest that the activity of TNT should be investigated in vivo to assess whether its potential hepatocarcinogenicity is realised in rats.
Animals↗
Comparisons between carcinogenic potency and mutagenic potency to Salmonella in a series of derivatives of 4-dimethylaminoazobenzene (DAB).
8 derivatives of the rodent liver carcinogen 4-dimethylaminoazobenzene (DAB), all of known carcinogenicity in rodents, have been evaluated in the 3 major variants of the Salmonella mutation assay; the standard plate test of Ames et al., the pre-incubation assay of Yahagi et al. and the fluctuation assay of Gatehouse. Although 4 of these chemicals were reported to be non-carcinogenic, and 4 to be of greater carcinogenic potency than DAB, each was mutagenic in a least 2 of the assays. Further, no quantitative correlation between carcinogenic and mutagenic potency was evident in any of the assay employed. The parent carcinogen DAB, 5-dimethylaminophenylazoindazole (a non-carcinogenic bacterial mutagen) and 6-dimethylaminophenylazobenzthiazole (a carcinogenic bacterial mutagen) were administered to rats via intraperitoneal injection, followed, 26 h later, by a sub-acute dose of [14C] dimethylnitrosamine. The histopathological condition of the livers of the treated animals was assessed together with a determination of the extent and nature of methylation by DMN of the DNA in the livers according to the method of O'Connor. Disturbances in both the pathological and DNA-related parameters were observed for the 2 carcinogens while control levels were seen for the non-carcinogen. Within this context the value of short-term assays conducted in vivo is discussed, especially their potential to identify potent mammalian carcinogens from among a collection of structurally related bacterial mutagens.
Animals↗
Synthesis of N7-hydroxyethylguanine and O6-hydroxyethylguanine. Markers for the reaction of ethylene oxide (EO) with DNA.
O6-Hydroxyethylguanine has been synthesized by reaction of mono-sodium glycolate with 6-chloroguanine. The crystalline product has been characterized using a variety of analytical techniques and compared with a sample of the corresponding N7-hydroxyethyl derivative. These 2 chemicals may prove useful as standards when studying the reaction of ethylene oxide (EO) with DNA.
Chemical Phenomena↗
Evaluation of two suggested methods of deactivating organic carcinogens by molecular modification.
Earlier observations that substitution of the aromatic nucleus of an arylamino/nitro carcinogen with either a sulphonic acid substituent or two methyl groups placed ortho to the nitrogen substituent renders the molecule non-carcinogenic have been extended via studies conducted in vitro. 4-Aminobiphenyl-4'-sulphonic acid has been synthesized and found to be non-mutagenic in the Salmonella mutation assay when tested under conditions where 4-aminobiphenyl was mutagenic. It is concluded that this sulphonic acid derivative may prove non-carcinogenic to rodents. In contrast to the non-carcinogenicity and non-mutagenicity reported for 3,3',5,5'-tetramethylbenzidine, 3,5-dimethyl-4-aminobiphenyl is approximately as mutagenic as 4-aminobiphenyl. It is therefore concluded that this material is potentially carcinogenic and that the loss of mutagenic activity observed for tetramethylbenzidine may be a structurally specific rather than a general phenomenon. In contrast, 3,5-dimethyl-4-nitrobiphenyl was much less mutagenic than 4-nitrobiphenyl. 9,9'-Bijulolidyl, a derivative of 3,3',5,5'-tetramethylbenzidine, was also found to be non-mutagenic. The general significance of these findings to the employment of structure-activity relationships in the design of non-mutagenic/non-carcinogenic molecules is discussed.
Aminobiphenyl Compounds↗
Norharman and ellipticine: a comparison of their albilities to interact with DNA in vitro.
The anti-tumor agent ellipticine has been compared in vitro with the bacterial co-mutagen norharman, a compound which it resembles superfically in chemical structure. Ellipticine was shown to stabilize the structure of double stranded calf-thymus DNA, to induce mutations in strain TA153 of Salmonella tryhimurium and to cause BHK cells to transform. Further, the major absorbance in its visible spectrum underwent a red shift of approximately 40 nm in the presence of native DNA. It is concluded that ellipticine intercalates with dna, and from this, that its action as an anti-tumor agent may, as has been previously suggested, be dependent upon this property. In contrast, norharman, a chemical suspected initially of being an intercalating agent, failed to stabilize the structure of DNA, was non-mutagenic to the same strain of S. typhimurium and was inactive as cell-transforming agent. In addition, its visible spectrum was not affected by the presence of DNA. The last observation is contrary to the conclusion of other workers, and an explanation of this difference is given. It is concluded that norharman is not capable of intercalating with DNA, and consequently, its mode of action as a co-mutagen is probably dependent upon its ability to inhibit certain mixed-function oxidase enzymes present in the liver activation system employed with in vitro mutagenicity assays.
Alkaloids↗
Some short-term tests for carcinogenesis.
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Animals↗