Search PubMed⌕ Search

Biomedical subjects

J Ashby

Publications and source records attributed to J Ashby.

At least 91 records · Page 5Linked to original sources
PubMed1997

Evaluation of the mutagenicity of acetochlor to male rat germ cells.

Male rat dominant lethal (DL) assays conducted on the herbicide acetochlor are described. Single dose studies conducted at the maximum tolerated dose (MTD, < or = 1000 mg/kg) produced no effects on any of the DL assay parameters at any of the ten weekly sampling periods. It is concluded that acetochlor is non-mutagenic to rat germ cells. Due to initial limited knowledge of the MTD of acetochlor it was also evaluated in the DL assay at a dose level of 2000 mg/kg. At this high dose level severe bodyweight loss and some deaths occurred among the treated animals. In addition, reduced implantations and reduced pregnancy rates were observed at the third sampling period (18-25 days post dosing) in the absence of an increase in early post-implantation deaths. These results indicated that the use of supra-MTD doses of acetochlor had reduced the fertility of the treated males leading to the production of a pseudo-DL assay response, as alerted to and defined by Ehling. Although several such pseudo-DL assay responses have been described, none have been explained mechanistically. It was therefore decided to pursue the effects seen in the DL assay when using supra-MTD doses of acetochlor. Ova analysis of female rats mated with male rats exposed to 2000 mg/kg acetochlor revealed unfertilized ova at the critical third sampling time. Normal fertilization of ova was observed at the first and fifth sampling period and, for a dose of 200 mg/kg acetochlor, at the third sampling period. The magnitude and temporal nature of these effects confirmed the induction of a pseudo-DL assay response, and studies were then undertaken to probe its genesis. Rats treated with 2000 mg/kg acetochlor had normal testicular and epididymal pathology and normal sperm numbers and sperm motility at the critical third sampling period. Despite a small reduction in testicular and epididymal glutathione levels 12 h after exposure to 2000 mg/kg acetochlor, testicular LDH and LDH-X enzyme levels were unaffected. Further, no reduction in the level of free sulphydryl groups (-SH) were observed in epididymal caput sperm heads isolated 0.5, 7 or 14 days after treatment of male rats with 2000 mg/kg acetochlor. The only sperm parameter affected by treatment with 2000 mg/kg acetochlor was an increase in epididymal cauda sperm with head abnormalities. The non-specific nature of this effect was considered inadequate to explain fully the high dose fertility effects seen in the DL assays, which therefore remain unexplained. The present data establish that acetochlor is non-mutagenic to rat germ cells. They also confirm the importance of segregating mutagenic and fertility effects in the DL assay, and emphasize the need for appropriate dose-setting studies prior to the conduct of rodent genetic toxicity assays.

Animals↗
PubMed1997

The genetic toxicity of time: importance of DNA-unwinding time to the outcome of single-cell gel electrophoresis assays.

Single-cell gel electrophoresis assays (comet assays) are described in which DNA damage is assessed in mouse skin keratinocytes treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL) either in vitro or in vivo. The positive results observed under both conditions of test encourage the further development of the mouse skin comet assay as a screen for direct-acting in vivo genotoxins. From the outset of the present experiments we were struck by the compacted nature of the DNA in mouse skin keratinocytes. Under similar conditions of assay, rodent hepatocytes presented a uniform 'unwound' distribution of DNA over the whole nuclear region. In order to study this effect we varied what seemed to be the most obviously related assay parameter: the DNA-unwinding time. A series of experiments was conducted in which control and MNNG-treated cells were exposed to a range of alkaline DNA-unwinding times (0.3-18 h) followed by measurement of the three comet tail parameters (length, DNA content, and their product, tail moment). Each of these parameters increased with increasing time of unwinding such that the tails observed for MNNG-treated cells with 0.3 h of DNA unwinding were similar in length to the tails of control cells exposed to an 8 h DNA-unwinding time. It is concluded that DNA-unwinding time is a critical parameter of the comet assay and that it may require optimisation for each tissue/cell type studied. Further, the data alert to the prospect that agents that uniquely affect chromosomal protein superstructure may increase comet tail length/DNA content in the absence of chemically induced DNA damage. Thus, there may be two discrete classes of chemical interaction with chromosomal DNA that yield identical comet assay results, but which have different implications for the genetic toxicity of the test agent. Similar effects were observed for rat hepatocytes or mouse lymphoma cells exposed to an 18 h DNA-unwinding time, but no comet tails were produced by exposure of cells to the lysis conditions (pH 10.0) for 18 h.

Acetone↗
PubMed1997

O6-benzylguanine potentiates the in vivo toxicity and clastogenicity of temozolomide and BCNU in mouse bone marrow.

The effects of treatment of mice with O6-benzylguanine (O6-BeG) on the levels of O6-alkylguanine-DNA alkyltransferase (ATase) in the hematopoietic compartment and on the in vivo sensitivity of hematopoietic progenitor cells to the toxic and clastogenic effects of the antitumor agents 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) and temozolomide were studied. When the overall effects of BCNU alone or with O6-BeG pretreatment were compared, dose potentiating factors of 4.17 for marrow cellularity, 4.57 for granulocyte macrophage-colony forming cells (GM-CFC) and 8.25 for colony forming unit-spleen (CFU-S) in O6-BeG pretreated versus nonpretreated animals were observed. A similar trend of dose potentiation was observed for temozolomide, although it was of lower magnitude: 1.20 for marrow cellularity, 1.63 for GM-CFC, and 1.68 for CFU-S. When the clastogenic effects of BCNU and temozolomide were examined in the mouse bone marrow micronucleus assay, a significantly (P < .05 to .001) higher frequency of micronuclei formation was observed in mice that received O6-BeG pretreatment compared with mice that received no pretreatment. These data suggest that the use of O6-BeG as a tumor-sensitizing agent before treatment of patients with O6-alkylating agents may lead to more severe hematological toxicity and possibly to an increased incidence of secondary leukemias as a result of elevated mutation frequencies in these patients.

Animals↗
PubMed1997

Mutation assays in male germ cells from transgenic mice: overview of study and conclusions.

Three confirmed mouse germ cell mutagens, ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS), have been evaluated for their activity as mutagens to the germ cell DNA of two strains of transgenic mice (lac I, Big Blue and LacZ, Muta Mouse). Both testicular DNA and epididymal sperm DNA were evaluated. A range of sampling times was studied, from 3 days post-dosing to 100 days post-dosing. ENU and iPMS were mutagenic to both testicular DNA and epididymal sperm DNA. Mutant frequencies were higher for both chemicals in DNA recovered from testicular tissue than in epididymal sperm DNA. Likewise, mutant frequencies were higher for both DNA samples at the later sampling times. MMS was not mutagenic under any condition of test. A good level of qualitative agreement in test results was seen for the two assays and for the same assays conducted in different laboratories. The level of quantitative agreement was not as high, but was, nonetheless, generally good. Recommendations for the future conduct of transgenic rodent germ cell mutation assays are made. The test data are discussed within the context of the larger question of how such assays should be integrated into the chemical hazard assessment process.

Animals↗
PubMed1997

The activity of ENU, iPMS and MMS in male mouse germ cells using the Muta Mouse positive selection transgenic mutation assay.

The alkylating agents ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS) are potent male rodent germ cell mutagens. The mutagenic activity of these compounds in male mouse germ cells has been evaluated using the Muta Mouse positive selection transgenic mutation assay. Both ENU (150 mg/kg) and iPMS (100 mg/kg) gave increased mutant frequencies in testicular DNA recovered 50 days after dosing. During the course of the mutation assays on iPMS its activity as a dominant lethal mutagen was confirmed by mating the treated animals with virgin (non-transgenic) females on day 10 post-dosing. Ova analysis on animals exposed to iPMS confirmed earlier reports that the dose level used caused sterility in mice 40 days after dosing. This sterility was shown to be due to aspermia in the treated mice at day 50 post-dosing. These collected findings indicate that at day 50 post-dosing with iPMS, mutations in testicular DNA can be observed in sterile animals. MMS (100 mg/kg) was not mutagenic to either testicular DNA or epididymal sperm DNA, 10 or 50 days, respectively, after dosing.

Animals↗
PubMed1997

The rodent uterotrophic assay: critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay.

The major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals. The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used, and the specificity of the assay for estrogens. It is concluded that three daily oral administrations of test chemicals to 21- to 22-day-old rats, followed by determination of absolute uterus weights on the fourth day, provide a sensitive and toxicologically relevant in vivo estrogenicity assay. Rats are favored over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor. Vaginal opening is shown to be a useful, but nondefinitive, adjunct to the uterotrophic assay. The ability of test chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay for the purpose of detecting antiestrogens. The results of a series of studies on the environmental estrogen nonyl phenol (NP), and its linear isomer n-nonyl phenol, confirm that branching of the aliphatic side chain is important for activity. 17beta-Desoxyestradiol is shown to be of similar activity to estradiol in the uterotrophic assay and is suggested to represent the "parent" estrogen of NP. Benzoylation of NP and 17-desoxyestradiol did not affect their uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol. Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay. Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence. However, the assay had a reduced ability (compared to rodents) to hydrolyze the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay. These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future. The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity.

Animals↗
PubMed1997

Activity of raloxifene in immature and ovariectomized rat uterotrophic assays.

Raloxifene is generally regarded as a tissue-selective estrogen agonist, being capable of selectively counter-acting both the loss of bone density and the increase in serum cholesterol that occur in rats following ovariectomy, without the induction of a trophic effect on the rat uterus. An implication of this activity profile is that reliance cannot be placed on the rat uterotrophic assay for the detection and assessment of xenobiotic estrogens. Within that context the estrogenic activity of raloxifene has been reevaluated in immature and ovariectomized rat uterotrophic assays. Four separate experiments were conducted. In the first two a reproducible increase (1.7-fold) was observed in the uterus wet weights of immature rats administered three daily doses of raloxifene. The maximum uterotrophic response observed over the dose range 0.01-2 mg/kg was for 0.1 mg/kg raloxifene. Further experiments utilized three daily doses of 0.1 mg/kg raloxifene. In the third experiment the uterotrophic response elicited by raloxifene in immature rats was abolished by coadministration of the estrogen receptor blocking agent Faslodex (ICI 182,780). This confirmed the direct involvement of estrogen receptors in the uterotrophic response elicited by raloxifene. Two further indications of the estrogenicity of raloxifene were obtained in this experiment. First, dry uterus weights were also shown to be increased by raloxifene administration, thereby eliminating water retention as the sole cause of the observed increases in uterus weights. Second, the height of the surface epithelium was increased by 1.7-fold in the raloxifene-treated animals, an effect that was accompanied by increases in mitotic activity and glandular formation in the stromal endometrium. The endometrial epithelium of the treated rats also showed evidence of vacuolation and, occasionally, the presence of degenerating cells. Raloxifene did not, however, cause premature vaginal opening in immature rats, unlike estradiol. In the fourth experiment the uterotrophic activity of raloxifene was confirmed in ovariectomized rats, although the response was less (1.2-fold) than in immature rats. In contrast to the effects seen for the positive control agent estradiol, the uterotrophic responses observed for raloxifene in ovariectomized animals were not accompanied by cornification of the vaginal epithelium. Premature vaginal opening and vaginal cornification may be less sensitive markers of estrogenic activity than the uterotrophic response. These collected observations confirm that raloxifene exerts a genuine trophic effect on the rat uterus, and as a consequence, the uterotrophic assay can be relied upon to detect estrogens with only a marginal effect on the uterus.

Administration, Oral↗
PubMed1997

Assessing the risks of adverse endocrine-mediated effects: where to from here?

This article is based on the closing lecture (by J. Ashby) of the meeting held in Research Triangle Park on January 13-14, 1997-"Assessing the Risks of Adverse Endocrine-Mediated Effects." Several summaries of unpublished data from Zeneca CTL are presented to illustrate concerns over the present absence of agreed criteria for the assessment of endocrine toxicity data. Reference is also made to several points raised by others during the course of the meeting. It is concluded that the substantial challenges currently faced can be met only by strict adherence to the basic principles of good scientific practice, preeminent among which are the needs to confirm data and to respond to them in a cool and dispassionate manner. The wealth of expertise and enthusiasm being devoted to the task in hand, by both governmental and industrial interests, suggests that practical means by which to address the topic of the symposium will be forthcoming in the near future. However, moves to accelerate progress unduly-"to conduct science under the gun"-are already evident. Therefore, if external influences demand that short-cuts must be taken toward achievement of the goals set out in the Wingspread Statement (Colborn and Clement, 1992), such compromises should be clearly identified in order not to confuse the underlying progress that is already evident in this new branch of toxicology.

Animals↗
PubMed1997

Failure to confirm estrogenic activity for benzoic acid and clofibrate: implications for lists of endocrine-disrupting agents.

Earlier reports that benzoic acid is uterotrophic to the rat and mouse and that clofibrate is uterotrophic to the rat have not been confirmed. The studies reported here involved the use of a range of test protocols and dose levels, including the protocols/dose levels used by the original investigators. In addition, both chemicals were inactive in a human estrogen receptor (hER alpha) yeast estrogenicity assay. It is concluded that benzoic acid and clofibrate are not estrogenic in the assays used here. This conclusion has implications for the compilation of lists of endocrine-disrupting chemicals.

Animals↗
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

The weanling male rat as an assay for endocrine disruption: preliminary observations.

Kelce and Wilson (J. Mol. Med. 75, 198-207, 1997) have suggested that dosing chemicals to newly weaned male rats for 1 month may yield a useful assay for antiandrogens. This suggestion was supported by reference to unpublished data on the antiandrogen vinclozolin which indicated reductions in the weight of accessory sex organs. The necessity for dosing during the full approximately 30 days of the protocol was not justified. An evaluation of this protocol has commenced by the dosing of vinclozolin, cyproterone acetate, and anastrozole daily to newly weaned male rats for 3, 7, or 14 days. No changes were observed in accessory sex organs when vinclozolin or anastrozole was dosed for 3 days. Significant changes were observed in the absolute and relative weight of all of the sex accessory organs for rats dosed for 7 or 14 days with cyproterone acetate. The effects produced by vinclozolin and anastrozole when dosed for 7 or 14 days varied according to the duration of exposure with the main effects on the accessory sex organs being seen after 14 days of dosing. The effects produced after 7 days of dosing with vinclozolin or anastrozole in arachis oil had resolved 10 days after the last of the seven doses. Data are presented using either hydroxy propyl methyoxycellulose (HPMC) or arachis oil as vehicle, the former being recommended for general use. These preliminary results are encouraging, and the evaluation of the second 2 weeks of the suggested 30-day protocol is proceeding. Concurrent control data indicate that the relative weight of the liver, testes, and epididymides increases over the first 14 days post-weaning, while those of the kidney, the seminal vesicles, and prostate decrease. These changes in relative tissue weight were much less than the increase in relative weight of the uterus observed in female animals at puberty. That indicates that successful use of a final version of this assay will depend on access to inhouse control tissue weight data and the use of appropriate animal group sizes. These preliminary data are presented to reduce duplication of effort in this rapidly expanding area of toxicology.

Anastrozole↗
PubMed1997

Review of the genotoxicity of 2-butoxyethanol.

The available data on the genotoxicity of 2-butoxyethanol have been reviewed. 2-Butoxyethanol has been examined for genotoxic activity in a range of in vitro and in vivo assays, including the mouse bone marrow micronucleus assay. The in vitro assays used range from well validated and generally accepted assays such as the Salmonella/microsome and in vitro cytogenetic assay, through to less well validated assays such as assessment of the inhibition of metabolic cooperation in V79 cells. The levels of experimental details and data reporting vary across the studies with some papers presenting only limited information. Taking the above factors into consideration, the available data indicate that 2-butoxyethanol has no significant genotoxic activity. This conclusion is also consistent with the chemical structure of 2-butoxyethanol, which is not alerting for likely genotoxic activity. These collected considerations indicate that 2-butoxyethanol is unlikely to be a genotoxic carcinogen to rodents, a prediction that supplements seventeen published predictions of the outcome of the ongoing NTP rodent carcinogenicity bioassays.

Acetaldehyde↗
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↗