An evaluation of caprolactam and benzoin in the mouse micronucleus test

Caprolactam (CAP) and benzoin (ZOIN) were tested in the mouse micronucleus test at two dose levels, one of which was the maximum tolerated dose. The compounds were administered by the oral route to groups of 5 male and 5 female mice. No statistical significant increase over control values of the frequency of PCE-containing micronuclei was observed at any dose level or sampling time, with the exception of CAP at a dose level of 700 mg/kg at the 24-h sampling time, where a small statistically significant effect was observed both when the sexes were analysed combined and separately. Due to this observation a limited repeat was carried out on CAP at the 700 mg/kg dose level at the 24-h sampling time. In the repeat study similar trends were observed even following the analysis of 5000 cells per animal. However, when these data were compared with historical control data no such effects were observed, the effects were therefore considered to be of questionable validity. Throughout the study the positive control (cyclophosphamide) gave an elevated biologically and statistically significant increase at all sampling times, thus verifying the sensitivity of the test system. It was therefore concluded that caprolactam and benzoin are not clastogenic in the mouse micronucleus test.     

Lack of DNA alterations induced by orotic acid in rat liver as evaluated with two DNA unwinding methods

One percent orotic acid supplemented diet is a promoting treatment in the rat model of liver carcinogenesis. After treatment with this type of diet, DNA alterations were observed using alkaline sucrose gradients and alkaline elution methods. In this work we have utilized two unwinding methods for the detection of DNA fragmentation. One method is a viscosimetric method in which the rate of increase in DNA viscosity with time is related to the rate of alkaline DNA unwinding. The second method measures fluorimetrically the amount of renatured and denatured DNA after different times allowed for alkaline DNA unwinding. These two methods are very sensitive in detecting DNA breaks induced by typical alkylating agents, X-rays and H2O2. The two unwinding methods were clearly negative for the orotic acid supplemented diet. We suggest that the DNA alterations detected with alkaline sucrose gradients and alkaline elution methods, after promoting treatment with orotic acid, are probably different from the DNA breaks induced by typical alkylating agents, X-rays and H2O2.     

Assessment of unscheduled DNA synthesis in Fischer 344 rat pachytene spermatocytes exposed to caprolactam or benzoin in vivo

Male Fischer 344 rats were treated with the non-carcinogenic chemicals CAP and ZOIN. The spermatogenic cells were isolated at selected times post-exposure for assessment of chemically-induced DNA damage by quantitative autoradiography of unscheduled DNA synthesis (UDS). Neither chemical (750 mg/kg administered by gavage) induced UDS in pachytene spermatocytes isolated 12, 24 or 48 h after treatment.     

The induction of DNA-strand breaks and unscheduled DNA synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin

Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 mg/kg and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled DNA synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment. These results correlate well with the observed lack of carcinogenicity of these compounds.     

DNA damage induced by some bile acids, evaluated with alkaline elution technique

Bile acids are promoting agents in colon carcinogenesis. In this work we have tried to characterize the DNA alteration induced by bile acids in Sprague-Dawley male rats. Confirming previous findings, a clear increase in elution rate was observed at alkaline pH. No effect could be observed when the nuclei were washed before the elution, in condition totally unsuitable for the repair of the type of DNA damage induced by typical genotoxic agents. We advanced the hypothesis that the increased alkaline elution rate observed with bile acids could be independent of DNA fragmentation and related to changes in chromatin structure.     

The effects of three non-antibiotic, antimicrobial agents on the surface hydrophobicity of certain micro-organisms evaluated by different methods

The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.     

Collagenase perfusion of rat liver induces DNA damage and DNA repair in hepatocytes

Evidence is presented that the collagenase perfusion of adult rat liver results in significant damage to nuclear DNA as evaluated by the alkaline elution technique. The extent of the damage is related to the perfusion time as well as to the clostridial enzyme preparation used. The DNA structure of isolated cells is almost completely repaired within 12 h of their culture in chemically defined medium.     

Chromosomal damage induced by caprolactam in human lymphocytes

Caprolactam was tested in the in vitro human lymphocyte cytogenetic assay both in the presence and absence of S9 mix at dose levels up to 5500 micrograms/ml using lymphocytes obtained from a male donor and in the presence of S9 mix using lymphocytes obtained from a female donor. Statistically significant increases in chromosomal damage were observed at 5500 micrograms/ml dose level in cells from both donors. This positive response was enhanced by the inclusion of chromosomal gaps in the calculations. It was concluded that caprolactam induces chromosomal damage in human lymphocytes in vitro albeit at comparatively high dose levels.     

The effects of three non-antibiotic, antimicrobial agents on the surface hydrophobicity of certain micro-organisms evaluated by different methods

D.S. JONES, S.P. GORMAN, D.F. MCCAFFERTY AND A.D. WOOLFSON. 1991. The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.     

Negative micronucleus tests on caprolactam and benzoin in ICR/JCL male mice

Male ICR/JCL mice were given a single intraperitoneal injection of 125, 250, and 500 mg/kg of caprolactam (CAP), or 250, 500, and 1000 mg/kg of benzoin (ZOIN). Bone marrow preparations were made 24, 30, and 48 h after treatment with the maximum dose, and 30 h after treatment with the other doses. The slides were coded before microscopic examination. No significant increase was found in the incidence of micronucleated polychromatic erythrocytes after treatment with either CAP or ZOIN.     

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron-induced DNA damage and synthesis in isolated rat liver nuclei.

Incubation of iron with isolated rat liver nuclei stimulated fragmentation of single-stranded DNA, incorporation of [3H]thymidine into DNA and the binding of 59Fe to DNA. FeCl2 was about twice as active as FeCl3. Lipid peroxidation took place in nuclei incubated with FeCl2, but not with FeCl3. Generation of reactive forms of oxygen was required for iron-mediated DNA damage, but evidence for direct interaction of reactive oxygen with DNA was not found. Apparent adducts of iron bound to DNA seemed to be formed by an enzymic mechanism.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain

The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated. Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU). The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH. Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH. The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight. The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA. With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h. The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period. The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.