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Biomedical subjects

H Tinwell

Publications and source records attributed to H Tinwell.

At least 55 records · Page 3Linked to original sources
PubMed1997

Normal sexual development of rats exposed to butyl benzyl phthalate from conception to weaning.

Butyl benzyl phthalate (BBP) has been administered in drinking water (1000 micrograms/liter) to pregnant AP rats during gestation and lactation. The sexual development of the pups was then monitored until their termination at postnatal day 90 (pnd 90). Pups derived from animals exposed to diethylstilbestrol (DES) in drinking water (50 micrograms/liter) acted as a positive control group. Glass drinking bottles were employed following demonstration of absorption of BBP into plastic drinking bottles. Drinking water intake was monitored and the estimated average exposures of the dams was 182.6 micrograms/kg/day BBP and 8.6 micrograms/kg/ day DES. A total of 240 control pups, 204 BBP pups, and 64 DES pups were obtained for study. The sexual development of both genders of pups was monitored. The body weights of the DES pups were significantly reduced at birth, an effect that persisted until pnd 90. The body weights of the BBP pups were marginally increased at birth, but this difference resolved by pnd 90. DES affected the sexual development of the pups for all endopoints monitored-anogenital (AG) distance on pnd 2; average day of vaginal opening and prepuce separation; uterus, testes, and accessory sex gland weight; and cauda epididymis sperm count and homogenization resistant testicular sperm count at pnd 90. BBP failed to affect any of these parameters, with the exception of a 1.1-day advance in the average day of vaginal opening and a small increase in male AG distance on pnd 2. These last two effects are related to the increased weight of the BBP pups. The incidence of FSH-containing cells in the pituitary gland of animals from each group was unaffected at pnd 90. The effects observed for the DES pups are consistent with the results of earlier studies by Sharpe et al. (Environ. Health Perspect. 103, 1136-1143, 1995). However, the absence of an effect of BBP administration on pup testis weight and testicular sperm count at pnd 90 is in contrast to reductions in these measurements reported earlier by Sharpe et al. (Environ. Health Perspect. 103, 1136-1143, 1995) for similarly derived BBP pups.

Animals↗
PubMed1997

Mutagenicity of the potent rat hepatocarcinogen 6BT to the liver of transgenic (lacI) rats: consideration of a reduced mutation assay protocol.

6-(p-dimethylaminophenylazo)benzothiazole (6BT) is an unusually potent rat hepatocarcinogen, producing large malignant liver tumours after only 2-3 months of dietary administration in a riboflavin-deficient diet. This azocarcinogen has been evaluated in a Big Blue F344 transgenic rat (lacI) gene mutation assay. In a reproduction of the early stages of the carcinogenesis bioassay of this agent, rats were maintained on a riboflavin-deficient diet and were given 10 consecutive daily doses of 6BT (10 mg/kg) by oral gavage. The animals were killed and the livers examined 11 days after the final dose. The livers of 6BT-treated rats showed evidence of hepatocellular hypertrophy in centrolobular areas, with some indication of an increased incidence of mitotic figures. An approximately 10-fold increase in the mutation frequency of DNA isolated from an aliquot of the combined liver homogenates of 6BT-treated rats was observed over that obtained from an equivalent aliquot from control animals. Examination of DNA samples isolated from the livers of individual animals confirmed that 6BT was mutagenic in Big Blue rat livers. These data extend the sensitivity of this transgenic assay to include azo hepatocarcinogens. The determination of mutation frequencies using pooled tissue samples represented a major resource-saving adaptation of the assay protocol in the present study; the general advantages and disadvantages of this practice are discussed.

Animals↗
PubMed1996

Mutagenicity to the mouse bone marrow by the mouse germ cell mutagen N-propyl-N-nitrosourea.

N-Propyl-N-nitrosourea (PNU) is shown to be active in male mouse bone marrow micronucleus assays when dosed at either 100 or 200 mg/kg in saline. Activity was observed following either intraperitoneal (i.p.) injection or oral gavage. This observation is consistent with the demonstration by Murota and Shibuya of the specific-locus mutagenicity caused by PNU in male mouse spermatogonia when dosed at 200 mg/kg by i.p. injection. These data strengthen further the observation that rodent germ cell mutagens are also mutagenic to rodent somatic cells.

Animals↗
PubMed1996

Fate of unfertilized ova in male rodent dominant lethal assays: extension of the studies by Kratochvilova.

Kratochvilova has described a technique whereby ova can be recovered from mated mice and their stage of division determined. This is of value to determine if reduced total implantations in a male dominant lethal (DL) germ cell mutation assay are due to pre-implantation loss of embryos, a presumed mutagenic event, or to chemically induced male infertility. Kratochvilova was not specific about the fate of unfertilized ova, but it was implied that they undergo a process of fragmentation that might be confused with the regular cleavage of fertilized ova. It became important for us to draw a firm distinction between ova fragmentation and regular ova cleavage in the rat. We therefore repeated the ova analyses of female mice mated with males exposed to iso-propyl methanesulphonate (iPMS), as described by Kratochvilova. Following that calibration study the technique was extended to the rat via ova cleavage analysis in mated female rats, coupled to a study of the normal decay of ova in virgin rats. Unfertilized ova are shown to undergo irregular fragmentation that can be clearly distinguished from normal cell division. It is concluded that the individual or combined incidences of single celled ova and fragmented ova (dependent on the cleavage stage of the concurrent control embryos) can provide a measure of male infertility as it relates to reduced implantations in DL assays. This ability to regard two morphological classifications of unfertilized ova as providing evidence for male infertility will simplify the conduct of ova analyses in both the mouse and the rat.

Animals↗
PubMed1996

The rodent bone marrow micronucleus assay: contrast between its sensitivity to human carcinogens and its insensitivity to NTP rodent carcinogens.

The rodent bone marrow micronucleus (MN) assay occupies a critical position in the accompanying schemes to detect potential human carcinogens and germ cell mutagens (Shelby, 1996; Ashby et al., 1996: for reviews of the MN assay see Heddle et al., 1983; Schlegel and MacGregor, 1984; CSGMT, 1990; Mavournin et al., 1990; Tinwell, 1990; Gatehouse, 1994; Asanami et al., 1995). The intention of this article is to note two perceptional problems currently associated with the MN assay. The first concerns how it should be used--as a screening assay, or as a means to evaluate the genetic toxicity in vivo of genotoxins defined in vitro. The second relates to its sensitivity to the rodent carcinogens defined by the US National Toxicology Program (NTP).

Animals↗
PubMed1996

Activity of iPMS and nPMS in mouse bone marrow micronucleus assays: comparison with mouse dominant lethal assay data.

isoPropyl methanesulphonate (iPMS) and its n-propyl analogue (nPMS) are shown to be active in mouse bone marrow micronucleus assays using male CBA, male and female (C3H/El X 102/E1)Fl and male and female Muta Mouse mice. iPMS was significantly more active than nPMS. No significant strain or gender differences were observed. These findings reflect the differences reported earlier for these two chemicals in mouse dominant lethal mutation assays. The earlier described dominant lethal assay data are represented schematically and discussed.

Alkylating Agents↗
PubMed1996

Unexpected genetic toxicity to rodents of the N',N'-dimethyl analogues of MNU and ENU.

Lijinsky and his colleagues have reported that the N',N'-dimethyl analogues of ENU and MNU [N',N'-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively] are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU. Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation. We therefore decided to study this problem further. Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH2 group with methyl groups (-NMe2) should ablate or abolish activity. Consistent with this DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration. Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively. Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the -NH2 group or by attack by water on the carbon of the carbonyl group. Methylation will inhibit both of these pathways, the first absolutely (no -NH2 protons) and the second partially, via steric inhibition. The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable. Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (approximately 300-fold increase in revertants at 2,000 micrograms/plate and approximately 180-fold increase in revertants at 150 micrograms/plate respectively). In contrast, the methylated analogues showed only weak mutagenic activity (approximately 3-fold) at approximately 100-fold higher dose-levels. Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU. To this point, hypothesis and data coincide. ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay. In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow. Four possible reasons for this conflict of theory and data are explored. The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals. With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis. Thus, only a small fraction of the administered dose survives to alkylate the bone marrow. Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow. However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment. If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained.

Alkylating Agents↗
PubMed1996

Evaluation of the potential carcinogenicity and genetic toxicity to humans of the herbicide acetochlor.

Comprehensive toxicological studies of the herbicide acetochlor are presented and discussed. Although it gave a negative profile of responses in the many toxicity tests conducted there were some findings that prompted further investigation. First, although non-mutagenic in the Salmonella assay, acetochlor was clastogenic to mammalian cells treated in vitro. This clastogenic potential was not expressed in vivo in four rodent cytogenetic assays (bone marrow and germ cells). Second, although acetochlor gave a negative response in rat liver UDS assays when tested at the acute MTD, gavage administration of a single, supra-MTD dose (2000 mg/kg) gave a weak positive assay response. This dose-level (2000 mg/kg) was necrotic to the liver, depressed hepatic glutathione levels by up to approximately 80%, altered the metabolism of acetochlor, and was associated with up to 33% lethality. In contrast, reference liver genotoxins such as DMN, DMH and 2AAF were shown to elicit UDS in the absence of such effects, and at approximately 400 x lower dose-levels. Finally, microscopic nasal polypoid adenomas were induced in the rat when acetochlor was administered for two years at the maximum tolerated dose (MTD). The tumours were not life-threatening, they did not metastasize, and no DNA damage was induced in the nasal cells of rats maintained on a diet containing the MTD of acetochlor for either 1 or 18 weeks (comet assay). In order to probe the mechanism of action of these high dose toxicities a series of chemical and genetic toxicity studies was conducted on acetochlor and a range of structural analogues. These revealed the chloroacetyl substructure to be the clastogenic species in vitro. Although relatively inert, this substituent is preferentially reactive to sulphydryl groupings, most evidently, to glutathione (GSH). Similar chemical reactivity and clastogenicity in vitro was observed for two related chemicals bearing a chloroacetyl group, both of which have been defined as non-carcinogens in studies reported by the US.NTP. These collective observations indicate that the source of the clastogenicity of acetochlor in vitro is also the source of its rapid detoxification in the rat in vivo, via reaction with GSH. Metabolic studies of acetochlor are described which reveal the formation of a series of GSH-associated biliary metabolites in the rat that were not produced in the mouse. The metabolism of acetochlor in the rat changes with increasing dose-levels, probably because of depletion of hepatic GSH. It is most likely that a rat-specific metabolite is responsible for the rat nasal tumours observed uniquely at elevated dose-levels. The absence of genetic toxicity to the nasal epithelium of rats exposed acutely or subchronically to acetochlor favours a non-genotoxic mechanism for the induction of these adenomas. The observation of a time- and dose-related increase in S-phase cells in the nasal epithelium is consistent with this conclusion. Despite some confusion caused by the early use of perilethal gavage administrations of acetochlor to rodents, and supra-MTD dietary concentrations in some of the chronic studies, the available MTD data are consistent with acetochlor not posing a genetic or carcinogenic hazard to humans.

Adenomatous Polyps↗
PubMed1995

Consideration of the liver of embryonic lacZ transgenic mice as an analogue of the mouse coat colour spot test: preliminary data and technical problems.

Pregnant lacZ+ transgenic mice (Muta Mouse) were treated with ENU (25 mg/kg) by oral gavage on day 10.5 of pregnancy. This dose of ENU is the optimal dose observed by other investigators for activity in the mouse coat colour spot test. Day 10.5 of pregnancy represents the stage when the embryonic liver first becomes visually discernable. By day 15.5, when the maternal and embryonic livers were analysed for lacZ- mutation frequency, the embryonic liver is the largest tissue in the embryo (approximately 25 mg). These experiments therefore represent treatment of a small pool of progenitor hepatocytes just as they are entering into an intense wave of cell division--optimum conditions for the fixation of mutations. Exposure to ENU led to an average fourfold, and a maximum tenfold increase in mutation frequency in the embryonic livers. This relatively weak response is consistent with lacZ- mutants not having a growth advantage; unlike in the mouse spot test, clonal amplification of lacZ- mutants cannot be separately scored. The level of mutation in the control embryonic livers was lower than that of the maternal control livers, but the group sizes were too small to conclude this definitively. Embryonic livers from each individual mother showed a range of mutation frequencies that were not obviously related to that of the maternal liver. On a treatment group basis, ENU was non-mutagenic to maternal livers. Half of the embryonic livers yielded DNA that failed to package, despite repeated attempts and re-isolation of the DNA from the liver. The cause of this unexpected finding is not clear. What is clear is that it was not due to the ENU treatment because eight of 22 control embryonic livers behaved similarly. These preliminary results suggest that further research is required in order to establish a practical transgenic analogue of the mouse coat colour spot test.

Animals↗
PubMed1995

A sequential approach to testing with the rodent bone marrow micronucleus assay--obviation of the need for statistical analyses of data.

The 'sequential' protocol we use for the conduct of mouse bone marrow micronucleus assays is described. The most important criterion for activity in the assay is the reproducibility of any induced effects. The frequencies of micronucleated polychromatic erythrocytes among 239 male CBA control mice are displayed and discussed. Our decision to avoid statistical analysis when assessing mouse bone marrow micronucleus data is explained.

Animals↗
PubMed1995

Comparison of lacI and lacZ transgenic mouse mutation assays: an EU-sponsored interlaboratory study.

As part of an EU Environment Programme sponsored study, the performance of two transgenic mouse mutation assays has been evaluated in three laboratories using common liver samples. The systems studied were the lacI-(Big Blue) mutation assay, and the GalE- positive selection modified lacZ- (Muta Mouse) assay. The liver samples compared were derived from mice treated with either saline or dimethylnitrosamine (DMN; 14 day recovery). Each assay gave an increased mutation frequency (MF) for the DMN-treated livers when compared to the saline control MFs. Sources of variability in the assays are discussed, and it is concluded that whole liver should be homogenised before DNA extraction, and that concurrent controls should be processed in parallel with test samples.

Animals↗
PubMed1995

The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment.

Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine (NDMA) for either 1 or 2 h and the cells assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay). A strong positive test response was observed producing comet tails of a length and DNA content not observed in either viable or dead control cells. A stronger test response was observed after a 2 h, as opposed to a 1 h, incubation of hepatocytes with NDMA. The method of processing the image of the comet is discussed and it is proposed that measurement of the length of the comet tail should commence at the estimated trailing edge of the cell, rather than at the leading edge or the estimated centre of the cell. Using this criterion, many control cells have no comet trails thereby enabling chemically induced tails to be more readily assessed. A simplified version of defining the comet tail moment is proposed.

Animals↗
PubMed1994

Mutation studies with dimethyl nitrosamine in young and old lac I transgenic mice.

Young adult (approximately 8 w) and old (approximately 72 w) lac I transgenic mice have been exposed to a single oral dose of dimethyl nitrosamine (NDMA). 2.5 h later unscheduled DNA synthesis was observed in the liver of the young animals (19.5 NG; control value -3.4 NG). The frequency of lac I- mutations in the liver of the young animals was elevated 7, 10 and 20 days after dosing. A similar fold-increase in mutant frequency was seen 7 days after dosing the old animals. It is concluded that mutability of the genome of old animals is not significantly different to that of young animals. Aspects of the minimum Big Blue assay protocol are discussed, as are the differing perceptions of the role of the assay in carcinogen hazard assessment.

Age Factors↗
PubMed1994

Response of the Muta mouse lacZ/galE- transgenic mutation assay to DMN: comparisons with the corresponding Big Blue (lacI) responses.

The lacZ Muta mouse transgenic mutation assay was recently adapted into a selective assay based on use of E. coli galE(-) bacteria and phenyl galactoside (p-gal). A preliminary assessment of this selective assay was undertaken using a single oral dose of 10 mg/kg of dimethyl nitrosamine (DMN). The livers of treated male mice were assessed for UDS 2 h after dosing, and for lacZ- mutations 7, 11 and 20 days after dosing. A strong UDS response was recorded and a clear mutagenic response was observed at each of the 3 timepoints. Comparison of these data with earlier data derived using the Big Blue (lacI) mutation assay reveals a marginally greater sensitivity to DMN of the selective Muta mouse assay, an effect probably related to a biochemical difference between the strains of animal, as evidenced by the larger UDS response seen in the Muta mouse system. The original Muta mouse assay protocol was impractical. The galE- adaption makes the assay emminently practical and cost-effective. We are continuing to assess the true role of both the Big Blue assay and the galE- Muta mouse assay in mutagenicity/carcinogenicity prediction. The former assay enables access to B6C3F1 mice and F344 rats, the latter enables the rapid acquisition of data.

Animals↗