The glutathione transferase Mu null genotype leads to lower 6-MMPR levels in patients treated with azathioprine but not with mercaptopurine.

The conversion of azathioprine (AZA) to mercaptopurine (MP) is mediated by glutathione transferase Mu1 (GSTM1), alpha1 (GSTA1) and alpha2 (GSTA2). We designed a case-control study with data from the TOPIC trial to explore the effects of genetic variation on steady state 6-methylmercaptopurine ribonucleotide (6-MMPR) and 6-thioguanine nucleotide (6-TGN) metabolite levels. We included 199 patients with inflammatory bowel disease (126 on AZA and 73 on MP). GSTM1-null genotype carriers on AZA had two-fold lower 6-MMPR levels than AZA users carrying one or two copies of GSTM1 (2239 (1006-4587) versus 4371 (1897-7369) pmol/8 × 108 RBCs; P<0.01). In patients on MP (control group) 6-MMPR levels were comparable (6195 (1551-10712) versus 6544 (1717-11600) pmol/8 × 108 RBCs; P=0.84). The 6-TGN levels were not affected by the GSTM1 genotype. The presence of genetic variants in GSTA1 and GSTA2 was not related to the 6-MMPR and 6-TGN levels.

Genetic polymorphism in UDP-glucuronosyltransferase 2B7 and colorectal cancer risk.

Colorectal cancer (CRC) is one of the most common malignancies in the Western world. CRC is strongly associated with lifestyle factors. Susceptibility to CRC may be partly due to deficient detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes result in variations in detoxification activities, which might influence the levels of carcinogens in the gastrointestinal tract, influencing the risk for CRC. To determine whether a genetic polymorphism in the detoxification enzyme UDP-glucuronosyltransferase 2B7 (UGT2B7) predisposes to CRC, 411 Caucasian patients with sporadic CRC and 600 Caucasian controls recruited from the same geographic area were genotyped for the functional UGT2B7 H268Y polymorphism. DNA was isolated and tested by a dual-color real-time polymerase chain reaction assay. Overall, no differences in genotype distributions between patients with CRC and controls were observed. When analyzed with respect to tumor location, a shift from the UGT2B7*I *2 into the UGT2B7*2*2 genotype was seen in patients with proximal CRC (OR 1.80, 95% CI 1.11-2.89). In the male patient subpopulation an even stronger association was observed (*1*1 + *1*2 vs. *2*2: OR 2.17, 95% CI 1.11-4.04; *1*2 vs. *2*2: OR 2.19, 95% CI 1.10-4.37). No associations with respect to tumor stage were seen. In conclusion, the frequency of the UGT2B7*2*2 genotype is higher in CRC patients with proximal location of the tumor, especially in males, which suggests that this genotype is associated with an increased risk for proximal CRC.

Detoxification enzyme polymorphisms are not involved in duodenal adenomatosis in familial adenomatous polyposis.

BACKGROUND: Patients with familial adenomatous polyposis (FAP) are at high risk of developing duodenal adenomas and carcinomas. Besides germline mutations in the adenomatous polyposis coli (APC) gene, additional factors may influence the age of onset and number of duodenal adenomas. This study compared the genotype distributions of duodenal detoxification enzyme isoforms in patients with FAP and controls. METHODS: The study included 85 patients with FAP and 218 healthy age- and sex-matched controls. Genotyping of all participants using polymerase chain reaction was performed to detect polymorphisms in isoforms of uridine 5'-diphosphate glucuronosyltransferases (UGTs) and glutathione S-transferases (GSTs): UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A10, UGT2B4, UGT2B7, UGT2B15, GSTA1, GSTP1, GSTM1 and GSTT1. RESULTS: The variant genotypes of UGT1A3 were less common in patients with FAP than in controls (odds ratio 0.39 (95 per cent confidence interval 0.22 to 0.67)). There were no associations between FAP and the other polymorphic genes. The polymorphisms investigated had no predictive value for the severity of duodenal adenomatosis in patients with FAP. CONCLUSION: Although the variant genotypes of UGT1A3 were less common in patients with FAP than in those without, this did not modulate the severity of duodenal adenomatosis.

Deficient UDP-glucuronosyltransferase detoxification enzyme activity in the small intestinal mucosa of patients with coeliac disease.

BACKGROUND: Small intestinal malignancies in humans are rare; however, patients with coeliac disease have a relatively high risk for such tumours. Intestinal UDP-glucuronosyltransferases are phase II drug metabolism enzymes also involved in the detoxification of ingested toxins and carcinogens. As many toxins and carcinogens are ingested via food, the human gastrointestinal tract not only has an important role in the uptake of essential nutrients, but also acts as a first barrier against such harmful constituents of the food. Therefore, the gastrointestinal mucosa contains high levels of detoxification enzymes such as cytochromes-P450, glutathione S-transferases and UDP-glucuronosyltransferases. AIM: To compare the UDP-glucuronosyltransferase detoxification capacity in small intestinal mucosa of patients with coeliac disease vs. that in normal controls. METHODS: We assessed UDP-glucuronosyltransferase enzyme activities towards 4-methylumbelliferone in small intestinal biopsies of patients with coeliac disease (n = 22) and age- and sex-matched controls (n = 27). RESULTS: Small intestinal UDP-glucuronosyltransferase enzyme activity in controls was significantly higher than in patients with coeliac disease: 0.55 +/- 0.27 vs. 0.35 +/- 0.16 nmol/min mg protein, respectively (mean +/- s.d., P = 0.005). DISCUSSION: The low small intestinal UDP-glucuronosyltransferase detoxification activity in patients with coeliac disease may result in a deficient detoxification of potential carcinogens, and thus could explain in part the relatively high small intestinal cancer risk in these patients.

Expression of the glutathione enzyme system of human colon mucosa by localisation, gender and age.

BACKGROUND: The glutathione S-transferases (GST) can metabolise endogenous and exogenous toxins and carcinogens by catalysing the conjugation of diverse electrophiles with reduced glutathione (GSH). Variations of GST enzyme activity could influence the susceptibility of developing cancers in certain areas of the gastrointestinal tract. AIMS: The expression of the components of the glutathione system in the colon was investigated with respect to age, gender and localisation. METHODS: Biopsies of macroscopically normal mucosa from both proximal and distal colon were collected from 208 patients (106 females, 102 males; mean age 61 years), who underwent colonoscopy for various clinical reasons. GSH content, total GST enzyme activity and the levels of the GST isoenzymes glutathione S-transferase P1 (GSTP1) and glutathione S-transferase M1 (GSTM1) were determined. RESULTS: GST enzyme activity, GSH and GSTP1 levels decreased significantly from proximal to distal colon (GST activity: 264 vs. 244 nmol/min/mg protein, p < 0.001, GSH content: 32 vs. 30 nmol/mg protein, p = 0.022 and GSTP1 levels: 2.25 vs. 2.10 mug/mg protein, p < 0.001). In female patients there was a significant stepwise increase of GST-activities and GSTP1 levels from the age of under 50 years to over 70 years. Oral sex hormone substitution among female patients between 50 and 70 years suppressed GST-activities and GSTP1 content. CONCLUSIONS: The GSH-system in the colonic mucosa is expressed at a lower level in the distal colon (sigma) than in the colon transversum; whether this small difference translates into variations of incidence of colorectal cancer remains to be seen. Females express higher enzyme levels as they grow older, while in males no significant age effects were found. Elderly females might be better equipped with protective GSH-enzymes in the colon than males and this could contribute to the lower incidence of colorectal carcinomas in females.

Biochemical characterization of resistance to mitoxantrone and adriamycin in Caco-2 human colon adenocarcinoma cells: a possible role for glutathione S-transferases.

Cytotoxicity of Adriamycin on human colon adenocarcinoma cell lines was investigated. Concentrations of Adriamycin producing 50% inhibition were very similar in HT29, Sw480, Sw620, and Sw1116 cells, whereas Caco-2 cells were relatively insensitive. As compared to the Sw1116 cell line, Caco-2 cells were also insensitive to mitoxantrone. Sensitivity to cisplatin, 5-fluorouracil, or ethacrynic acid was comparable in both cell lines. To find the mechanism for this mitoxantrone and Adriamycin resistance, several potential Adriamycin-detoxifying systems were characterized and quantified in both Sw1116 and Caco-2 cells. No dramatic differences in glutathione content and expression of both selenium dependent- and independent glutathione peroxidase, UDP-glucuronyltransferase, and cytochrome P-450 were found. However, highly significant differences in glutathione S-transferase activity were present, the expression of both class pi and class alpha glutathione S-transferases being much higher in the Caco-2 cell line. In addition, a slightly higher content of P-170 glycoprotein was present in the Caco-2 cells. These findings suggest that glutathione S-transferases, and to a lesser extent the P-170 glycoprotein, may be involved in mitoxantrone and Adriamycin resistance of Caco-2 colon carcinoma cells.

Glutathione S-transferase pi in colorectal tumors is predictive for overall survival.

Glutathione S-transferases (GSTs) are enzymes involved in the detoxification of xenobiotics and are divided into four subclasses, alpha, mu, pi, and theta, with different although overlapping substrate specificities. Most human gastrointestinal tumors contain increased amounts of GST-pi and GST enzyme activity. The relationship between GST parameters and tumor and patient characteristics, including overall survival, were studied retrospectively in 100 primary colorectal adenocarcinomas. Levels of GST-alpha, GST-mu, GST-pi, and GST enzyme activity were not related to the Dukes stage, differentiation grade, localization, histological type and diameter of the tumor, or gender and age of the patient. Fifty-seven patients died (median survival, 21 months; range, 1-65 months) during follow-up, and 43 patients were still alive at the closing date of the study (median follow-up, 68 months; range, 60-87 months). Optimal dichotomization and uni- and multivariate analyses were done with the Cox proportional hazard model. Multivariate analysis with all clinicopathological parameters revealed higher Dukes stage (hazard ratio, 2.7; P < 0.001) and older age (hazard ratio, 2.8; P = 0.001) to be the only independent prognostic variables for overall survival. In contrast to GST-alpha and GST-mu, high levels of GST-pi (hazard ratio, 3.1; P = 0.002) and GST enzyme activity (hazard ratio, 2.0; P = 0.020) in the tumors were found to have a significant prognostic value independent from the clinicopathological parameters when added separately to this Cox model. Thus, this study indicates that GST subclass levels in colorectal adenocarcinomas are not related to clinicopathological parameters and that the GST-pi level and GST enzyme activity have a prognostic value for the overall survival of the patients.

Polymorphic expression of the glutathione S-transferase P1 gene and its susceptibility to Barrett's esophagus and esophageal carcinoma.

Factors determining individual susceptibility to esophageal cancer or premalignant Barrett's epithelium are still largely unclear. An imbalance between phase I drug metabolism [e.g., cytochrome P450 (CYP)] and phase II detoxification [e.g., glutathione S-transferase (GST)] may contribute to the development of these diseases. Polymorphic variants in the CYP1A1 gene were described leading to increased levels of bioactive compounds, whereas polymorphisms in GST genes often resulted in impaired detoxification. We studied the frequencies of polymorphic variants in CYP1A1, GSTP1, GSTT1, and GSTM1 genes in 98 patients with Barrett's epithelium and 34 patients with esophageal cancer. The results were compared with those obtained from 247 healthy blood donors. DNA was extracted, and PCR-RFLP methods were used to detect genetic polymorphisms. Chi2 analysis, Spearman rank correlation, and Wilcoxon rank sum tests were used for statistical evaluation. Polymorphisms in CYP1A1, GSTM1, and GSTT1 occurred at an equal frequency in patients and controls. Occurrence of the polymorphic GSTP1b variant in the GSTP1 gene resulted in a significantly lower GST enzyme activity (P < 0.05), and GSTP1b was found significantly more often in patients with Barrett's epithelium (70%; P < 0.001) and patients with esophageal adenocarcinoma (76%; P = 0.005), as compared to healthy blood donors (41%). In conclusion, presence of the GSTP1b allele leads to lower GST enzyme activity levels and, consequently, impaired detoxification. This most important esophageal GST isoform may, therefore, contribute to the development of Barrett's epithelium and adenocarcinoma.

Nonsteroidal anti-inflammatory drugs enhance glutathione S-transferase theta levels in rat colon.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been claimed to reduce cancer rates in oesophagus, stomach and colon of humans and laboratory animals. Recently we showed that dietary administration of NSAIDs enhanced glutathione S-transferase (GST) class alpha, mu and pi levels in the upper part of the rat gastrointestinal tract, with minor effects in the colon. Enhancement of GSTs, a family of detoxification enzymes consisting of class alpha, mu, pi and theta isoforms, might be one of the mechanisms leading to cancer prevention. The recently cloned GST class theta levels have not yet been studied in this respect. We now investigated whether the NSAIDs indomethacin, relafen, sulindac, ibuprofen, piroxicam, and acetyl salicylic acid (ASA), incorporated individually into the diet at 25, 200, 320, 400, 400 and 400 mg/kg, respectively, affect gastrointestinal GSTT1-1 and GSTT2-2 levels in male Wistar rats. GSTT1-1 and GSTT2-2 levels were determined in cytosolic fractions of oesophagus, gastric, small intestinal and colonic mucosa and liver by densitometrical analyses of Western blots after immunodetection with a monoclonal (GSTT1-1) or a polyclonal (GSTT2-2) antibody. Gastric GSTT2-2 levels were induced by ibuprofen (1.6x) and indomethacin (1.5x), and colonic levels were induced by ASA (1.7x). Colonic GSTT1-1 levels were elevated by all NSAIDs tested except for relafen (1.5-6.4x). In conclusion, enhancement of colonic GSTT1-1 levels seems to be a common working mechanism of NSAIDs. Enhanced enzyme activity, which may result from these higher GSTT1-1 levels, might lead to a more efficient detoxification of potential carcinogens and hence contribute to the prevention of colon carcinogenesis.

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes.

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.

Fibrinogen and albumin synthesis are regulated at the transcriptional level during the acute phase response.

During acute inflammation or after administration of monocytic products, an enhanced transcription of the fibrinogen polypeptide genes and a reduced transcription of the albumin gene were observed. The changes in the fibrinogen polypeptide transcriptional rate were found to precede the change in albumin gene transcription. These findings indicate that the altered synthesis of fibrinogen and albumin during inflammation are regulated at the transcriptional level and are most probably mediated by monocytic products (including interleukin-1).

Urinary GSTP1-1 excretion is markedly increased in normotensive pregnancy as well as in preeclampsia.

BACKGROUND: Glutathione S-transferases (GSTs) are present in large amounts in the human kidney, where they demonstrate a specific distribution. The assessment of urinary excretion of GST alpha (proximal tubules) and pi (distal and collecting tubules) could be helpful in determining if, and to what degree renal tubular damage is present in preeclampsia and whether this damage is in the proximal or distal region. METHODS: Urine samples were collected from 22 women with severe preeclampsia and/or HELLP syndrome (PE), from 30 non-pregnant women with a history of severe preeclampsia (HPE), from 18 women with uncomplicated pregnancies (PC) and from 30 non-pregnant women with a history of uncomplicated pregnancies (HPC). GSTA1-1 and GSTP1-1 were assayed by ELISA and were expressed as nanograms per 10 mmol creatinine (Cr). RESULTS: Median urinary GSTP1-1 concentrations were significantly (p<0.001) higher in women with preeclampsia [62.2 (4.3-291.2) ng/10 mmol Cr] compared to non-pregnant women with a history of preeclampsia [22.3 (0-142.6) ng/10 mmol Cr]). In addition, in normotensive pregnant women, urinary GSTP1-1 concentrations were significantly (p<0.01) higher [82.6 (8.3-206.7) ng/10 mmol Cr]) compared to non-pregnant controls [5.1 (0-66.7) ng/10 mmol Cr]. No difference in GSTP1-1 concentrations was found between women with preeclampsia and normotensive pregnant women. GSTA1-1 concentrations were not significantly different between the four groups of women investigated. There were no correlations between the degree of proteinuria and urinary GSTP1-1 or GSTA1-1 concentrations. CONCLUSION: GSTP1-1 metabolism in the distal tubule changes during normotensive as well as preeclamptic pregnancy. Whether this is due to tubular cell damage, disturbed resorption or an increase in cellular levels cannot be determined as yet.

A polymorphism in the gene for microsomal epoxide hydrolase is associated with pre-eclampsia.

OBJECTIVE: Microsomal epoxide hydrolase is an important enzyme involved in the metabolism of endogenous and exogenous toxicants. Polymorphic variants of the human epoxide hydrolase gene vary in enzyme activity. We determined whether genetic variability in the gene encoding for microsomal epoxide hydrolase contributes to individual differences in susceptibility to the development of pre-eclampsia with or without the syndrome of Haemolysis, Elevated Liver enzymes, and Low Platelets (HELLP). METHODS: A total of 183 non-pregnant women with a history of pre-eclampsia, 96 of whom had concurrently developed the HELLP syndrome, and 151 healthy female controls were genotyped for the 113Tyr-->His polymorphism in exon 3 and the 139His-->Arg polymorphism in exon 4 of the epoxide hydrolase gene by a polymerase chain reaction-restriction fragment length polymorphism assay. Chi-square analysis was used for statistical evaluation of differences in polymorphic rates. RESULTS: In pre-eclampsia a higher frequency (29%) of the high activity genotype Tyr113 Tyr113 in exon 3 was found as compared to controls (16%, OR 2.0, 95% CI 1.2-3.7). There was no difference between groups for the 139His-->Arg polymorphism. In women with a history of pre-eclampsia, no difference in epoxide hydrolase genotypes was found between women who either did or did not develop the HELLP syndrome. In addition, a significant association was found between predicted EPHX activity and pre-eclampsia. CONCLUSIONS: Women with the high activity genotype in exon 3, which could reflect differences in metabolic activation of endogenous or exogenous toxic compounds, may have enhanced susceptibility to pre-eclampsia. However, polymorphisms in the epoxide hydrolase gene do not seem to influence the risk for concurrent development of the HELLP syndrome.

Mutagenicity of five arylamines after metabolic activation with isolated dog and human hepatocytes.

The mutagenicity of benzidine (BZ) N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 4-aminobiphenyl (4-AB) and 2-aminoanthracene (2-AA) towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from dog and man. The influence of paraoxon, an inhibitor of the deacetylation reaction, on the mutagenicity of these compounds was also investigated. Obvious interspecies differences in the mutagenic activation of benzidine and its acetylated-derivatives were seen. However, with liver cell preparations from both species it was found that MABZ and DABZ were more mutagenic than BZ itself. 4-AB appeared to be weakly mutagenic in the presence of human hepatocytes but non-mutagenic with dog hepatocytes. 2-AA was highly mutagenic in both species. When human hepatocytes were used as the metabolic factor, the mutagenicity of all arylamines decreased in the presence of paraoxon. With dog hepatocytes, however, the mutagenicity of all arylamines except DABZ was enhanced in the presence of paraoxon.

Mutagenicity and DNA-excision repair induced by isoniazid after metabolic activation by isolated human and rat hepatocytes.

The mutagenic potency of isoniazid (INH) (a widely used antitubercular drug) towards Salmonella typhimurium strain hisG46 was studied in the Salmonella/hepatocyte suspension-assay, comprising isolated human or rat hepatocytes as metabolic system. The potency of INH to induce DNA-excision repair in these hepatocytes was also measured. With rat hepatocytes, INH appeared to be only weakly mutagenic and did not induce significant increases in hepatocellular DNA-excision repair. With isolated hepatocytes of two human subjects, INH appeared also only weakly mutagenic. However, with hepatocytes of two other human subjects, INH was found to be highly mutagenic. Comparable results were obtained for the induction of hepatocellular DNA-excision repair.

Effects of treatment with the opioid antagonists naloxone and naltrexone on LH secretion and on sexual maturation in immature female rats.

The effects on sexual maturation of the opioid receptor antagonists naloxone and naltrexone were studied in the female rat. Neonatal treatment (days 1-10) with either naloxone (2.5 mg/kg at 6-h intervals) or naltrexone (20 or 50 mg/kg per day) did not advance sexual maturation as judged by age and body weight at vaginal opening and first ovulation. After treatment with naltrexone (20 mg/kg) first ovulation occurred 2.3 days earlier than in saline-treated control rats but this could be attributed to a growth-stimulating effect of naltrexone; the effect was not observed with 50 mg/kg. An effect of neonatal treatment with naloxone on serum LH levels was seen at 23 days of age (155 +/- 36 (S.E.M.) vs 14 +/- 4 micrograms LH/l in controls, P less than 0.01), but not at 29 or 33 days of age, at 2 days before first ovulation nor at first pro-oestrus. There were no differences in the number of ova released at first oestrus, nor in the length of the first cycle. Neonatal treatment with naltrexone (50 mg/kg per day) did not alter the response to treatment with human chorionic gonadotrophin at 28-31 days of age: ovulation of a mean of 7.3 ova was induced on day 32 in both naltrexone- and saline-treated rats. Naltrexone treatment (four daily injections of 20 mg/kg at 2-h intervals) at 28-32 days of age advanced first ovulation by 4.4 days in about 40% of the rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturation of the inhibitory feedback action of oestrogen on follicle-stimulating hormone secretion in the immature female rat: a role for alpha-foetoprotein.

The maturation of the inhibitory feedback action of oestrogen on FSH secretion in the immature female rat was studied from 5 days of age until after the first ovulation. To study the role of the oestrogen binding alpha-foetoprotein (AFP) which is present in the blood of young animals, the effects of various doses of oestradiol and of the synthetic oestrogen R2858 (11 beta-methoxy-17-ethynyl-oestradiol), which is not bound by AFP, were compared in ovariectomized rats. A rise in the serum concentration of FSH within 2 days of ovariectomy was first observed in rats ovariectomized at 8 days of age. Between 8 and 28 days of age the rise in FSH after ovariectomy could be prevented by oestrogen injections in such a way that the resulting FSH concentration amounted to 50% of that in ovariectomized control rats. This was achieved with a constant dose of 0.00015 microgram R2858/100 g body weight, whereas the dose of oestradiol needed decreased from 0.05 to 0.01 microgram/100 g body weight indicating an increased sensitivity to the feedback action of oestradiol. After day 28, sensitivity to the feedback action of both R2858 and oestradiol decreased progressively up to the time of the first ovulation. In contrast to results at earlier ages, none of the doses of either oestrogen was capable of maintaining near-physiological concentrations of FSH after 20 days of age. It is concluded that the apparent increase in sensitivity to the feedback action of oestradiol occurring before 28 days of age reflects the disappearance of AFP from the blood, whereas the subsequent decrease in sensitivity is independent of AFP. Moreover, it is concluded that up to about 20 days of age oestradiol could be, though not necessarily is, the sole ovarian factor involved in regulating FSH secretion, whereas at later ages additional steroids and/or factors must be involved.

Retardation of first ovulation in pubertal rats after treatment with 5 alpha-androstane-3 alpha, 17 beta-diol or its 3 beta-epimer.

The effect was studied of five daily injections of 50 microgram of either 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol) or its 3 beta-epimer (3 beta-androstanediol), starting on day 22 of life, on sexual maturation in female rats. No difference was found in the age and body weight at first oestrus between oil-treated rats and rats treated with either 3 alpha- or 3 beta-androstanediol. The only difference observed between these groups consisted of the occurrence of a "pinhole" vaginal opening a few days before oestrus in 50% of the steroid-treated rats; in oil-treated rats immediate full vaginal opening and first oestrus coincided in ten out of 12 rats. Different effects were obtained when the higher dose of 100 microgram daily was used; effects were dissimilar in rats treated with 3 alpha- and 3 beta-androstanediol. If administration of the higher dose of 3 beta-androstanediol was started on day 22 and continued until the day of full vaginal opening and first oestrus, a significant delay of this first oestrus, preceded by a few days of a "pinhole" type of vaginal opening was observed. After administration of the higher dose of 3 alpha-androstanediol a "pinhole" type of vaginal opening, accompanied by dioestrous-like vaginal smears, was also found, but oestrus did not occur during the period when injections were given. After the injections were stopped on day 45, first oestrus developed within 6 days in all rats. The previous findings of others that administration of 3 beta-androstanediol to the immature female rat may induce precocious puberty (i.e. precocious vaginal opening and first ovulation) were not confirmed in the present study. Out results indicate that high doses of free 3 alpha-androstanediol, and to a lesser degree 3 beta-androstanediol, may even delay first ovulation in the rat. A possible interference of 3 alpha-androstanediol with the triggering of the first ovulatory gonadotrophin peaks is discussed.

Effects of adrenalectomy on the release of follicle-stimulating hormone and the onset of puberty in female rats.

The involvement of the adrenal gland in the release of gonadotrophins and the onset of puberty in female rats was studied. Two and four days after adrenalectomy (ADX) on either day 5 or 10 after birth, a significant decrease in the concentration of FSH was found; 4 days after ADX on either day 15 or 20, FSH concentrations had increased significantly compared with sham-operated and/or intact controls. However, in the rats adrenalectomized on day 15 or 20, the body weights were lower than in control rats. Relative uterine weights (mg/100 g body wt) in adrenalectomized rats never differed from those of control rats. A delay in the time at which vaginal opening and the first oestrus occurred was found in rats adrenalectomized at 20 or 25 days of age; however this delay was accompanied in these rats by a retardation in the gain in body weight. It is argued that the effects of ADX on both the release of gonadotrophins and the onset of puberty are primarily, and presumably exclusively, due to the effects on general bodily development (expressed in body weight). The lack of effect of ADX on uterine weight supports the hypothesis that 'oestrogen-like' products from the adrenal gland are not biologically active as oestrogens.

Effects of sodium pentobarbitone administration on gonadotrophin release, first ovulation and ovarian morphology in pubertal rats.

Pubertal female rats received sodium pentobarbitone (PB; 45 mg/kg body wt) at various hours on the day of first pro-oestrus. Maximal blockade of ovulation, in about 60% of the rats, occurred after PB treatment at 12.00, 13.00 and 14.00 h. The number of small antral follicles (100-499 X 10(5) mum3) was reduced 1 day after PB treatment in both blocked and ovulating rats. In the ovaries of non-ovulating rats signs of stimulation by LH such as dispersion of cumulus cells, oocyte maturation and early luteinization were sometimes present; in ovulating rats cystic corpora lutea with entrapped ova were found in addition to normal corpora lutea. Gonadotrophin measurements after PB treatment (14.00 h) in pubertal and adult rats showed (at 17.00 h) reduced levels of both LH and FSH, these levels being lower in the adults. Gonadotrophin levels of blocked and ovulating pubertal rats overlapped. In PB-treated, pubertal rats in which ovulation was postponed by 1 day, vaginal oestrus was prolonged by 1 day and the subsequent dioestrus by 2 days. The pubertal rat is thus less sensitive to PB treatment than the adult. PB treatment of the younger animal influences not only the ovulatory process but also follicular growth and, presumably, the length of the approaching cycle.