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J A Vericat

Publications and source records attributed to J A Vericat.

At least 19 recordsLinked to original sources
PubMed2003

A new hepatoma cell line for toxicity testing at repeated doses.

Many cell models that are used to assess basic cytotoxicity show a good correlation with acute toxicity. However, their correlation with the toxicity seen following chronic in vivo exposure is less evident. The new human hepatoma cell line HBG BC2 possesses the capacity of being reversibly differentiated in vitro and of maintaining a relatively higher metabolic rate when in the differentiated state (3 weeks) as compared to HepG2 cells, and thus may allow the conduct of repeated toxicity testing on cells in culture. In order to evaluate the genetic background of HBG BC2 cells, the expression of selected genes was analyzed in untreated cultures and, in addition, the behavior of HBG BC2 cultures under conditions of repeated treatment was studied with acetaminophen as a test substance and coupled with the use of standard staining techniques to demonstrate toxicity. Results showed that cultures of HBG BC2 cells retained a capacity to undergo apoptosis and proliferation, allowing probable replacement of damaged cells in the culture monolayer. MTT reduction was used to evaluate the toxicity of acetaminophen, acetylsalicylic acid, perhexiline, and propranolol, after both single and repeated (3 times/week for 2 weeks) administration. Under the conditions of repeated treatment, cytotoxicity was observed at lower doses as compared to single administration. In addition, the lowest nontoxic doses were in the same range as plasma concentrations measured in humans under therapeutic use. Our results suggest that the new human hepatoma HBG BC2 cell line is of interest for the evaluation of cell toxicity under conditions of repeated administration.

Acetaminophen↗
PubMed1999

Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse bone marrow micronucleus test.

Ten halogenated aliphatic hydrocarbons (carbon tetrachloride, 1-chlorohexane, 2,3-dichlorobutane, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, hexachloroethane, 1,1,2-trichloroethane, 1,2,3-trichloropropane and 1,1,3-trichloropropene), previously assayed in genetic assays in fungi, were evaluated in the mouse bone marrow micronucleus test in order to assess their genotoxicity in vivo. All chemicals were administered once i.p. at 40 and 70-80% of their respective LD50 to male and female CD-1 mice, 24 and 48 h before killing. All treatments produced evident clinical symptoms, but no marked depression of bone marrow proliferation. No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed. The comparison of the results obtained in this study with the findings provided by in vitro micronucleus assays on the same chemicals, reported by other authors, indicate that mouse bone marrow is weakly sensitive to the genotoxic effects induced by halogenated hydrocarbons in other test systems. This suggests that the role of such an assay in carcinogen screening may be questionable for this chemical class. An examination of mouse bone marrow micronucleus test results with the halogenated aliphatic hydrocarbons classified as carcinogens by IARC supports this conclusion.

Animals↗
PubMed1997

Genotoxicity studies on ebrotidine.

Five genotoxicity studies on ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio] ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542), including at least four of the battery of tests recommended by toxicological regulatory guidelines for new drugs, were conducted. These tests were the Ames test for determination of bacterial gene mutations, sex-linked recessive lethal mutation test in Drosophila for gene mutations in eukaryotic systems, in vitro chromosome aberration test and micronucleus test for evaluation of structural and numerical aberrations, and sister chromatid exchange frequency test for assessment of non-specific damage to chromatin. Negative and positive controls were used in all the experiments. The effects were investigated in the absence or presence of metabolic activation by S-9 microsomal fraction from rat liver homogenate. A dose range toxicity study was also performed to determine the dosage levels or concentrations to be tested for the assessment of genotoxic effects. None of the tests showed a significant increase in the genotoxic parameters, both in vitro and in vivo in somatic or germ cells. It is, therefore, concluded that ebrotidine has not caused mutagenic or clastogenic effects in any of the experimental systems tested.

Animals↗
PubMed1996

Light-dependent activation of 7,12-dimethylbenz[a]anthracene to a potent genotoxicant.

7,12-Dimethylbenz[a]anthracene (DMBA), which is widely used in mutagenesis and experimental carcinogenesis, is activated to a mutagen by white fluorescent light. A 40 min exposure to white fluorescent light of Salmonella typhimurium TA98 plates treated with DMBA, in the absence of exogenous metabolism, resulted in an approximately 30-fold increase in the number of histidine revertants. This phenomenon also occurs, with lesser intensity, with other promutagens, such as benzo[a]pyrene or 2-acetylaminofluorene, and in other Salmonella tester strains. Moreover, white fluorescent light is able to activate DMBA to a toxicant for Chinese hamster V79 cells in culture, resulting in very low cell survival. Under these conditions, white fluorescent light-activated DMBA was shown to cause chromosomal aberrations, but not gene mutations, as determined by resistance to thioguanine. This white fluorescent light-dependent activation of DMBA seems to be related to the formation of reactive species, as the addition of vitamin E results in a reduction in the number of histidine revertants induced by white fluorescent light in S. typhimurium TA98.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Absolute configuration of 7,12-dimethylbenz[a]anthracene-DNA adducts in mouse epidermis.

32P-Postlabeling was used to monitor the formation of DMBA-DNA adducts in mouse epidermis from each enantiomer of the trans 3,4-dihydrodiol. It was shown that the (4R,3R)-dihydrodiol is converted to the anti (4R,3S)-dihydrodiol (2S,1R)-epoxide which reacts with deoxyguanosine and deoxyadenosine residues in epidermal DNA to yield two of the major adducts formed when DMBA itself binds to epidermal DNA. The third major DMBA-derived adduct with deoxyadenosine residues was shown to arise from the (4S,3R)-dihydrodiol through the intermediacy of the syn (4S,3R)-dihydrodiol (2S,1R)-epoxide.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Alternative methods in toxicology tests: in vitro toxicity.

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develop alternative methods to reduce the required number of animals. The new in vitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by the in vitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity with in vitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems for in vitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mitochondrial activity, intracellular content and leakage of enzymes into the medium.

Animal Testing Alternatives↗
PubMed1989

Absolute stereochemistry of the major 7,12-dimethylbenz[alpha]anthracene- DNA adducts formed in mouse cells.

In recent work we assigned partial structures to individual 7,12-dimethylbenz[alpha]anthracene (DMBA)--deoxyribonucleoside bisphosphates separated by TLC after postlabeling with [32P]ATP. We have now been able to postlabel DNA adducts formed in cells exposed to either the (4R,3R)- or (4S,3S)-dihydrodiol of DMBA and thereby to assign absolute stereochemistry to the 2-, 3- and 4- positions in the major DMBA-DNA adducts. It is found that the major anti dihydrodiol epoxide-DNA adducts arise from the (4R,3S)-dihydrodiol (2S,1R)-epoxide and that the major syn dihydrodiol epoxide-DNA adducts arise from the (4S,3R)-dihydrodiol (2S,1R)-epoxide.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells.

UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.

DNA Repair↗
PubMed1988

Adaptive response to simple alkylating agents in the phototrophic bacteria Rhodobacter capsulatus and R.sphaeroides.

The presence of an adaptive response to low doses of alkylating chemicals in the phototrophic bacteria Rhodobacter sphaeroides and R.capsulatus has been studied. Results obtained show that both strains display this repair response against the challenge doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), when they are pretreated with low doses of this compound for 120 min. The adaptive response of both R.sphaeroides and R.capsulatus induced an increase of cell survival and a decrease of mutagenesis in the MNNG-pretreated cells. Furthermore, the MNNG-induced adaptive repair also gives protection to diethylsulphate and ethylmethanesulphonate in both phototrophic bacteria. Finally, the MNNG-promoted adaptive response is sensitive to inhibitors of protein synthesis such as chloramphenicol, indicating that this DNA repair mechanism needs protein synthesis in R.sphaeroides and R.capsulatus, in a way similar to that which occurs in Escherichia coli.

Chloramphenicol↗
PubMed1988

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.

Alkylation↗
PubMed1986

Effect of alkylating agents on the expression of inducible genes of Escherichia coli.

Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli. This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.

Alkylating Agents↗
PubMed1985

Further characterization of SOS system induction in recBC mutants of Escherichia coli.

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC- mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC- cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC- mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC- and wild-type strains.

Adenine↗
PubMed1985

Expression of the SOS genes of Escherichia coli in Salmonella typhimurium.

To lysogenize Salmonella typhimurium by Lambda phage, a region of 10.2 kb of Escherichia coli DNA carrying the nusA gene was cloned in a S. typhimurium strain containing a F'112 plasmid which codifies for the lamB region of E. coli. The strain of S. typhimurium obtained in this way, was lysogenized by lambda c IndO- bacteriophage harboring either a fusion between recA1 or sfiA genes of E. coli with lacZ gene. Likewise, pSE143 plasmid with a umu C::lacZ fusion was introduced in S. typhimurium. Afterwards, induction of these SOS genes was studied. Results obtained show that the basal transcription of both recA and sfiA genes of E. coli was higher in S. typhimurium than in E. coli. Nevertheless, induction of recA and sfiA genes by UV-irradiation and mitomycin C was higher in E. coli than in S. typhimurium. On the other hand, umuC gene of E. coli presents the same basal level of transcription in both E. coli and S. typhimurium species, although induction of this gene by UV-irradiation and mitomycin C was higher in S. typhimurium than in E. coli. Therefore, the plasmid pUA25 constructed in this work may be used to introduce, using the Lambda phage as a vector, the SOS genes of E. coli in other bacterial species which may be useful to study the relationship between their respective SOS systems.

Bacterial Proteins↗
PubMed1984

Effect of L-ethionine on the expression of the SOS system in Escherichia coli.

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine, on the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA+ Met+ RelA+ strain, nor in RecA+ Met- RelA+ or RecA+ Met- RelA- mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42 degrees C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42 degrees C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA+ strain even if this compound is added to the cells several hours before irradiation.

DNA Repair↗
PubMed1983

Discriminated induction of SOS functions in Escherichia coli by alkylating agents.

Treatment of Escherichia coli with the alkylating agents diethyl sulphate, ethyl methane-sulphonate and N-methyl-N'-nitrosoguanidine produces a different pattern of expression of SOS functions. There is a full induction of recA-dependent inhibition of cell respiration, a slight induction of lambda prophage, and no inhibition of cellular division. In a comparative study with bleomycin, an agent which is able to induce these three SOS functions, we have also shown that the differences in expression of SOS functions are not due to any variation in the pattern of DNA synthesis, or DNA degradation after treatment with alkylating agents. These results suggest that the kind of damage induced in the DNA may be important in determining which SOS function is expressed.

Alkylating Agents↗
PubMedDate not supplied

Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli.

Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division. This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition. The same result is obtained when a culture of E. coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine. The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose. Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C. This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process. These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced.

Antibiotics, Antineoplastic↗