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H Tinwell

Publications and source records attributed to H Tinwell.

At least 19 recordsLinked to original sources
PubMed2004

Testosterone-stimulated weanlings as an alternative to castrated male rats in the Hershberger anti-androgen assay.

We showed previously that stimulation of weanling male rats with the synthetic androgen 17-methyltestosterone (17MT) caused premature growth of the sex accessory tissues such that the activity of the two anti-androgens flutamide and DDE could be demonstrated (Regul. Toxicol. Pharmacol. 35 (2002) 280). We suggested that that protocol should be evaluated as an alternative to the castrated male rat Hershberger assay. In the present paper we justify changing the assay protocol to use testosterone propionate (TP), in place of 17MT, as the stimulating androgen. This change enables biochemical formation of dihydrotestosterone from testosterone, a conversion not possible when using 17MT. This change in the protocol enables detection of the testosterone-5-reductase inhibitor finasteride. The modified TP-stimulated weanling male rat assay is shown to have similar sensitivity to that of the castrated male rat Hershberger assay in detection of the anti-androgens flutamide, procymidone, vinclozolin, and DDE, and of the biochemical inhibitor finasteride. The anti-androgen linuron and the anabolic steroid trenbolone were also detected as positive by the TP-stimulated weanling male assay. It is suggested that this modified assay for anti-androgens should be validated as an alternative to the Hershberger assay, thereby reducing animal stress by obviating the need for surgical castration.

Androgen Antagonists↗
PubMed2003

The effect on sperm production in adult Sprague-Dawley rats exposed by gavage to bisphenol A between postnatal days 91-97.

M. Sakaue et al. (2001,J. Occup. Health vol. 43, pp. 185-190) have described how oral exposure of sexually mature male rats to bisphenol A (BPA) between postnatal days (PND) 91-97 led to a reduction in daily sperm production (DSP) 5 weeks later (18 weeks of age). Activity was observed over the dose range 20 microgram/kg-200 mg/kg BPA, with an absence of activity over the dose range 2 ng/kg-2 microgram/kg BPA. There was no evidence of a dose response relationship over the active dose range (five orders of magnitude range). The observation of endocrine disruption (ED) effects for BPA at such low doses, and in sexually mature animals, was unexpected. It was therefore decided to mount an independent repeat of their study. A total of four independent studies were conducted according to the protocol used by Sakaue et al. Doses of 20 microgram/kg, 2 mg/kg, or 200 mg/kg BPA were administered to adult Sprague-Dawley (SD) rats over PND 91-97, and the studies were terminated when the rats reached the age of 18 weeks. Three different rodent diets were employed (RM3, Purina 5002, and CE2), the last of which had been used by Sakaue et al. BPA failed to give any evidence of ED activities, including the changes in DSP reported by Sakaue et al. 2001. During the course of these studies, the test protocol was adapted to coincide more precisely with that used by Sakaue et al.; this included restricting the number of animals per cage, removing bedding from the cages, and changing to the use of glass water bottles in the cages. The only thing of interest to emerge from our studies was the observation of a significant difference in DSP between the control groups of our first and second study. As the change in diet from RM3 to Purina 5002 was the major difference between those two studies, we conducted a repeat of the second study, but we were unable to confirm the differences seen between the first and second study. The probability that those differences arose either by chance, or as the result of intrinsic study-to-study variability, was strengthened by the absence of significant differences in the sperm parameters in a final (fifth) study where the sperm parameters for control animals maintained on the three different diets were compared under the conditions of the main experiments. No explanation for our failure to replicate the effects reported by Sakaue et al. is evident. A review of DSP values reported in the recent literature is provided and discussed, and it is concluded that use of the term DSP/g testis rather than DSP/testis could increase the sensitivity of DSP assessments.

Animals↗
PubMed2002

The effects of atrazine on the sexual maturation of female rats.

The mammalian hazard assessment of the herbicide atrazine (ATR) has focused on the induction of mammary tumors and accelerated reproductive aging of adult rats, and the relationship of these effects to the inhibition of leutinizing hormone (LH) release from the pituitary, an effect itself caused by inhibition of GnRH signaling by the adult rat hypothalamus. In earlier studies, Laws et al. (Toxicol. Sci., 58, 366-376, 2000) demonstrated a delay in female rat sexual maturation induced by ATR, effects that could equally have been caused by inhibition of hypothalamic GnRH release. The present studies were designed to compare the doses that interfere with GnRH signaling seen in previous studies in adult Sprague-Dawley (SD) rats (LH surge suppression) with doses that impair GnRH signaling in peripubertal rats, as indicated by delayed sexual maturation. The studies evaluated the effects of ATR treatment on the timing of uterine growth and vaginal opening (VO) in peripubertal female Wistar (Alderley Park, AP) and SD rats. Doses of 10, 30, and 100 mg/kg ATR were administered daily from postnatal day (pnd) 21 to up to pnd 46. Determinations of uterine weight were made at pnd 30, 33, 43 (AP), and 46 (SD) and the timing of VO was also assessed in the last two of these experiments. The centrally acting GnRH antagonist Antarelix (ANT) was used as a positive control agent as it has previously been shown to prevent uterine growth and to delay VO in peripubertal AP rats. Uterine growth and VO were completely prevented in AP rats exposed to ANT. Uterine growth was delayed at pnd 30 and 33 in AP rats exposed to 100 mg/kg ATR, but this growth inhibition had been overcome by pnd 43. VO was significantly delayed in AP rats for the 100 mg/kg ATR dose. By pnd 46, VO was significantly delayed in SD rats exposed to both 30 and 100 mg/kg ATR, but uterine weights were unaffected by that time (as for AP rats). It is concluded that the no-effect level for the effects of ATR on sexually immature rats (10 mg/kg in SD; 30 mg/kg AP) is approximately the same as reported previously by Laws et al. in peripubertal Wistar rats (25 mg/kg). However, the no-effect level in peripubertal female SD rats is nearly an order of magnitude greater than the no-observed effect level observed in female SD rats fed ATR for 6 months (1.8 mg/kg) where LH suppression was used as an indicator of effect on the pituitary/hypothalamic axis (USEPA, Atrazine-DACT Fourth Report of the Hazard Identification and Review Committee, April 5, 2002). These results support the conclusion that the pituitary/hypothalamic axis in peripubertal female SD rats is less sensitive than that in adult female SD rats.

Administration, Oral↗
PubMed2002

Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning.

The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures. The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data. In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 microg DES/l drinking water ( approximately 6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero, and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1-21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero. No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1-10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero, or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning. This study therefore does not establish a unique role for exposures in utero or during the early neonatal period.

Abnormalities, Drug-Induced↗
PubMed2002

Normal sexual development of two strains of rat exposed in utero to low doses of bisphenol A.

Pregnant Sprague-Dawley (SD) and Alderley Park (Wistar derived) rats were exposed by gavage during gestation days 6-21 to 20 microg/kg, 100 microg/kg, or 50 mg/kg body weight of BPA with ethinylestradiol (EE; 200 microg/kg) acting as a positive control agent. The sexual development of the derived pups was monitored until termination at postnatal day 90-98. The endpoints evaluated were litter size and weight, anogenital distance at birth, days of vaginal opening, first estrus and prepuce separation, weights of the liver, seminal vesicles, epididimydes, testes, ventral prostate, uterus, vagina, cervix and ovaries, and daily sperm production. Males were terminated at postnatal day 90 and females at postnatal day 98. The only statistically significant effects observed for any dose of BPA were a decrease in daily sperm production and an increase in the age of vaginal opening for the Alderley Park animals at the highest dose evaluated (50 mg/kg). The dose of EE evaluated proved to be maternally toxic in our laboratory, but provided gross evidence of endocrine disruption in the treated dams. These results diverge from those of Chahoud and his colleagues who indicated disturbances to the sexual development of both male and female SD rat pups administered the same 3 doses of BPA. This failure to confirm low dose endocrine effects for BPA is discussed within the context of similar divergent conclusions derived from other assessments of the endocrine toxicity of this agent to rats.

Animals↗
PubMed2001

Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

Adenine Phosphoribosyltransferase↗
PubMed2001

The uterotrophic activity of nonylphenol in the rat is not mediated by aromatase enzyme induction.

p-Nonylphenol (NP) is weakly estrogenic to rodents and to some species of fish. All evidence to date has indicated that the estrogenic effects of NP are due to the interaction of NP with the estrogen receptor. Recent findings of increased plasma estradiol in fish exposed to NP have, however, led to the proposal of an alternative mechanism for NP-induced estrogenicity in this species, possibly via induction of aromatase enzymes. In the present studies, this hypothesis was investigated in rats using the aromatase inhibitor anastrozole. The results indicated that the uterotrophic action of NP, as with estradiol used as a positive control, is mediated directly by its interaction with uterine ER, rather than an indirect effect via aromatase enzyme induction. Circulating levels of estradiol were unchanged after NP treatment and the aromatase inhibitor anastrozole failed to inhibit NP-induced uterine growth. These results are consistent with previous published data on NP in rodents.

Anastrozole↗
PubMed2001

Lack of binding to isolated estrogen or androgen receptors, and inactivity in the immature rat uterotrophic assay, of the ultraviolet sunscreen filters Tinosorb M-active and Tinosorb S.

The presence of structurally diverse chemicals as contaminants in the environment has led to concerns regarding their possible endocrine disturbing effects. Recently, some ultraviolet absorbing components of sunscreen preparations have given positive responses in assays monitoring estrogen-like activity both in vitro and in vivo. Consequently, two recently developed sunscreen components, Tinosorb M-active and Tinosorb S, were evaluated using the in vitro estrogen and androgen receptor competitive binding assays. Neither compound gave a positive response in either of the assays, consistent with the large molecular dimensions of each chemical disfavoring binding to the hormone receptors. Both of the chemicals were inactive in immature rat uterotrophic assays conducted using the subcutaneous route of administration. It is concluded that neither of these agents possess intrinsic estrogenic/antiestrogenic or androgenic/antiandrogenic activity. The several positive control chemicals evaluated gave the expected positive responses in the assays used.

Animals↗
PubMed2001

The application of the restriction site mutation assay to compare 1-ethyl-1-nitrosourea-induced mutations between the endogenous p53 gene and the transgenic LacZ gene in MutaMouse testes.

Transgenic mouse modelling has provided a new approach to study the various steps involved in spontaneous and induced mutagenesis in rodent somatic and germline tissues in vivo. However, the important question arises as to whether mutations occur at the same rate in transgenes as in endogenous genes. Here, the restriction site mutation (RSM) assay was used to study mutations induced in the endogenous p53 gene and LacZ transgene of MutaMouse testes treated with 1-ethyl-1-nitrosourea (ENU). The aim of these experiments was to compare mutation susceptibility between the endogenous p53 gene and the integrated LacZ gene in the transgenic mouse. ENU-treated and control testes were analysed 102 days after treatment; a total of 297 RSM analyses were performed on ENU-treated and untreated testis DNA. Ten mutational events were detected in the p53 gene (exon 5 and intron 8), two of which occurred in untreated animals and probably represent spontaneous events. Only a single mutation was detected in the LacZ gene of an ENU-treated animal by the RSM assay. Thus the RSM assay can readily detect ENU-induced mutations in the p53 gene, but not in the LacZ transgene. Comparison of the LacZ RSM mutation data with results from a previous study of identically dosed MutaMice in the transgenic selection assay [Ashby, J., Gorelick,N.J. and Shelby,M.D. (1997) Mutat. Res., 388, 111-122] showed that LacZ mutations were far more readily recovered with the MutaMouse transgenic selection assay than by RSM analysis. The reason for the relative inability of the RSM assay to detect LacZ mutations may be the smaller target size of the RSM analysis compared with the transgenic selection assay (16 bases compared with 3000 bases). Taking into account the different target sizes by calculating the mutation frequency per base allowed the RSM data regarding p53 and LacZ to be compared with previously published data from transgenic selection assays. These studies demonstrated that the p53 mutations were present at mutation frequencies (per base) 5- to 70-fold higher than the LacZ gene mutations. In addition, the LacZ mutation frequency per base found in the RSM was an order of magnitude higher than that found in the transgenic selection assay. The transgenic selection assay is more sensitive per locus (due to the larger target of the LacZ gene), as evidenced by ability to detect ENU-induced testes mutations readily.

Alkylating Agents↗
PubMed2001

The male rat carcinogens limonene and sodium saccharin are not mutagenic to male Big Blue rats.

Limonene and sodium saccharin are male rat specific carcinogens giving rise to renal and bladder tumours, respectively. Both compounds give negative results in genetic toxicity assays suggesting a non-genotoxic mode of action for their carcinogenicity. The alpha 2U-globulin accumulation theory has been invoked to explain the renal carcinogenicity of limonene: the accumulation of micro masses of calcium phosphate in the bladder, coupled with a high pH environment in the male rat bladder, has been suggested to be responsible for the bladder carcinogenicity of sodium saccharin. The implication of these proposed mechanisms is that limonene and sodium saccharin will not be mutagenic to the rat kidney and bladder, respectively. This proposal has been evaluated by assessing the mutagenic potential of the two chemicals to male lacI transgenic (Big Blue) rats. Male Big Blue rats were exposed for 10 consecutive days to either limonene in diet, at a dose level in excess of that used in the original National Toxicology Program gavage carcinogenicity bioassay, or to sodium saccharin in diet at the dose known to induce bladder tumours. The multi-site rat carcinogen 4-aminobiphenyl was used as a positive control for the experiment. Limonene failed to increase the mutant frequency in the liver or kidney of the rats, and sodium saccharin failed to increase the mutant frequency in the liver or bladder of the rats. 4-Aminobiphenyl was mutagenic to all three of these tissues. These results add further support to a non-genotoxic mechanism of carcinogenic action for both limonene and sodium saccharin.

Administration, Oral↗
PubMed2001

Effect of rodent diets on the sexual development of the rat.

Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined. Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist ANTARELIX: The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating the potential endocrine toxicity of chemicals, the components of rodent diets used should be known, and as far as is possible, controlled.

Age Factors↗
PubMed2000

Uterotrophic activity of bisphenol A in the immature mouse.

Bisphenol A (BPA) has been evaluated in eight independent immature mouse uterotrophic assays using the subcutaneous route of administration, and in a single study employing oral gavage. The dose range covered was from 0.02 microg to 300 mg/kg BPA and some experiments were supplemented by assessments of uterine hypertrophy and hyperplasia. Pooling of the test data indicates no uterotrophic activity for the chemical. However, in a subset of the subcutaneous injection studies, where control uterine weights were relatively low, significant, but weak, uterotrophic activity was observed over a range of dose levels, but in the complete absence of a dose relationship. In the oral gavage study, no increases in uterine weight were seen, but there were increases in uterine labeling with bromodeoxyuridine at 200-300 mg/kg BPA. The present study illustrated that when a large number of observations are made, a certain level of chance observations may be made, and that surrogates for an increase in uterine weight do not necessarily enhance assay sensitivity, albeit such data may complement uterine weight data. The data indicate that reducing control uterine weights may enhance assay sensitivity, but that animal body weight is an imperfect indicator of control uterine weight. The data also show that it is possible for individual investigators to be unable to confirm their own observations. It is concluded that BPA may be weakly uterotrophic to the mouse under specific conditions of test, and in the complete absence of a dose-response relationship to this activity. However, overall, we have failed to define BPA as reproducibly active in the immature mouse uterotrophic assay, and in that sense, our data are broadly consistent with those reported earlier by Coldham et al. (Environ. Health Perspect. 105, 734-742, 1997) in 1997 using a similar assay.

Administration, Oral↗
PubMed2000

The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

ATP Binding Cassette Transporter, Subfamily B, Mem↗