Evaluation of genotoxic potential of norethynodrel in human lymphocytes in vitro.

The genotoxicity study of a synthetic progestin norethynodrel, was carried out on human lymphocytes chromosomes using sister chromatid exchanges (SCEs), replication index (RI) and chromosomal aberrations (CAs) as parameters. The study was carried out in the presence and absence of metabolic activation (S9 mix). Norethynodrel was studied at three different concentrations (20, 40 and 60 microg/ml of peripheral blood lymphocyte culture) and was found non-genotoxic in the absence of metabolic activation. But in the presence of S9 mix norethynodrel increased SCE (p<0.03) and CA (p<0.005) frequencies and inhibits lymphocyte proliferation (p<0.03) at 60 microg/ml. The results suggest a genotoxic and cytotoxic effect of norethynodrel in human lymphocytes in vitro in the presence of S9 mix.

An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio.

The aim of the present study was to elucidate how full life-cycle exposure to estrogens impacts zebrafish development and reproduction, compared to partial life-cycle exposure only, and whether the estrogen-induced effects in zebrafish are reversible or irreversible. Zebrafish were exposed in a flow-through system to an environmentally relevant concentration (3 ng/L) of the synthetic estrogen 17alpha-ethinylestradiol (EE2) either from fertilization until the all-ovary stage of gonad development (i.e., 42 d postfertilization [DPF] in our experiment) or from fertilization until the reproductive stage (i.e., 118 DPF). Reversibility of the estrogen-induced effects was assessed after 58 d of depuration in EE2-free water until 176 DPE Early life exposure led to a lasting induction of plasma vitellogenin (VTG) in adult females but altered neither the sex ratio nor the reproductive capabilities. Full life-cycle exposure resulted in elevated VTG concentrations and caused gonadal feminization in 100% of exposed fish and thus inhibited reproduction. Two types of ovaries were observed in continuously exposed adult fish, immature ovaries with primary growth stage oocytes only and mature ovaries containing the full range of all oocyte maturation stages. Fish with immature ovaries had plasma VTG levels like control males, while fish with mature ovaries had female-like VTG levels. The effects of full life cycle exposure were at least partly reversible, and 26% of fish of the previous all-female cohort developed fully differentiated testes. These findings suggest that continuous estrogen exposure had arrested the developmental transition of the gonads of genetic males from the early all-ovary stage to functional testes. After the exposure had ceased, however, these males apparently were able to accomplish testicular differentiation.

Potentiation of the vitellogenic response to 17alpha-ethinylestradiol by cortisol in the fathead minnow Pimephales promelas.

The effects of elevated plasma cortisol levels on vitellogenin (VTG) induction were examined in the fathead minnow (Pimephales promelas) in an attempt to evaluate the potential influence of stress on this commonly used biomarker of estrogenicity. Two separate experiments were conducted in which fish plasma cortisol was elevated to various levels for 14 d by noninvasive additions of cortisol to the aquaria water. Fathead minnows were exposed to either cortisol alone, 17alpha-ethinylestradiol (EE2) alone, or a combination of the two hormones, and plasma levels of VTG as well as liver expression of VTG mRNA were measured. Both experiments gave similar results, with an exposure to 4 ng/L of EE2 resulting in significantly greater levels of plasma VTG in the presence of, compared to that in absence of, cortisol, whereas exposure to cortisol alone at concentrations between 144 and 800 microg/L had no effect on plasma VTG levels. This potentiation of the EE2-induced vitellogenesis by cortisol was dose-dependent, with plasma VTG reaching 125, 167, and 295% of the values obtained with EE2 alone when 144, 360, and 800 microg/L of cortisol, respectively, were added to the water. Liver mRNA results were consistent with plasma VTG, although they generally were more variable. The present study demonstrates that cortisol does not independently induce vitellogenesis but can potentiate estrogen-induced VTG synthesis in fathead minnow. The implications of these findings for the use of VTG as a biomarker of estrogenicity are discussed.

Chlamydial salpingitis in female guinea pigs receiving oral contraceptives.

Female guinea pigs were given daily doses of a combination of oral contraceptive (OC) agents, consisting of mestranol and norethynodrel suspended in sesame oil or distilled H2O, and were infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC). Counts of chlamydial inclusions in cells of vaginal smears collected during infection, showed prolongation and enhancement of infection in OC-treated animals as compared with controls. Appearance of IgG and IgA antibodies to GPIC in genital secretions, as determined by enzyme-linked immunosorbent assay (ELISA), was also delayed in OC-treated animals as compared with controls. OC-treated infected animals were killed on days 15 and 43, and gross pathological evidence for ascending infection culminating in salpingitis was found in all of five and four of five animals, respectively. On the other hand, among untreated infected controls on each sacrifice day, only one of five animals had any evidence for ascending infection. Chlamydiae were detected by light and electron microscopy in fallopian tube tissue collected on day 15 following OC-treatment but not in tissue from control animals.

Alteration of convulsive threshold and conditioned avoidance response in mice fed diets containing contraceptive steroids.

Female CD1 mice given the contraceptive steroids mestranol and norethynodrel (1:10) in the diet (0.0033%) for 4 months had a growth reduction of 20% when compared with mice fed a normal diet, and had lower convulsive thresholds when tested with the vitamin B6 antagonists 2,4-dimethyl-5-methyl-hydroxypyrimidine and thiosemicarbazide. In conditioned avoidance response (CAR) tests, mice fed the steroid-containing diets showed a decreased acquisition performance during all six sessions; however, mice fed the same diet supplemented with vitamin B6 (0.04%) performed as well during the last three sessions of the CAR tests as mice fed the normal diet.

Enhancement of mutagenic activity in Salmonella by contraceptive steroids.

Two oral contraceptive steroids, mestranol and norethynodrel, were evaluated for mutagenicity in the Salmonella histidine reversion assay. The pure forms of the hormones were not mutagenic when tested with either missense (TA1535, TA100) or frameshift (TA98, TA1538, TA1537) strains. In vitro activation of the hormones with liver homogenates from rats induced either with phenobarbital or Aroclor did not influence these results. However, mestranol was capable of enhancing the mutation yield obtained by an ineffective subthreshold dose of 2-acetylaminofluorene. Dimethyl sulfoxide extracts of two contraceptive pills, Ovulen-21 (containing mestranol) or Enovid-E (containing mestranol or norethynodrel), also were nonmutagenic. But again, both these extracts were capable of enhancing the mutation yield induced with an ineffective dosage of 2-acetylaminofluorene and N-nitrosopiperidine. These studies point to the possible promotional effect and subsequent potential hazard to the female consumers who use these hormones as a means of pregnancy control.

Assay of some endogenous and synthetic sex steroids for tumor-initiating activity in rat liver using the Solt-Farber system.

Endogenous (estradiol-17 beta; progesterone) and synthetic (ethinyl estradiol; cyproterone acetate; norethindrone acetate; norethynodrel) sex steroids were evaluated for tumor-initiating activity in the rat liver using the Solt-Farber system. All steroids were negative. This provides further evidence that tumor formation in long-term rodent bioassays by these compounds may be due to epigenetic mechanisms.

Lack of hepatogenotoxicity of oral contraceptive steroids.

The objective of the present study was to determine whether mestranol could initiate hepatocarcinogenesis and whether various gonadal steroids have detectable hepatogenotoxic potential. To test for initiation potential, female, Sprague-Dawley rats were partially hepatectomized and intubated with mestranol (100 or 500 mg/kg) 24 h later. Twenty-four hours after treatment all rats were transferred to diet containing 0.05% phenobarbital to promote hepatocytes initiated by mestranol treatment. Four months later an analysis of putative preneoplastic gamma glutamyl transpeptidase positive foci indicated that mestranol did not cause a significant increase in the number of such foci. Hepatogenotoxicity was assessed in two ways, first using alkaline elution to detect liver DNA damage after in vivo treatment with mestranol, and second by detection of DNA repair synthesis in primary cultures of hepatocytes treated with various oral contraceptive steroids. The results of both studies were negative. In conclusion, the oral contraceptive steroids do not exhibit strong initiating or genotoxic potential.

Norethynodrel.

PIP: This monograph on norethynodrel (NOR) includes chemical and physical data (synonyms and trade names), structural and molecular formulae and molecular weight of NOR, chemical and physical properties of the pure substance (e.g., melting point, optical rotation, and solubility), and the production, use, occurrence, and analysis of the drug. NOR is produced by reducing estradiol 3-methyl ether with lithium in liquid ammonia followed by ethynolation which produces the 3-methyl ether of NOR; treatment with acetic acid realized NOR. NOR is not known to occur naturally. It is used primarily as a progestin in oral contraceptives combined with estrogens. It is also used to treat dysfunctional uterine bleeding and endometriosis. The agent sees greater commerical use in Europe than in the U.S. Analytical methods for determining the bulk chemical are presented tabularly. Biological data relevant to the evaluation of carcinogenic risk in humans are presented in brief. NOR has been tested in rats, mice, and monkeys, alone and combined with estrogens, via various delivery routes. Alone, NOR increased incidences of pituitary tumors in mice of both sexes and of mammary tumors in castrated male mice of 1 strain. Male rats showed increases in liver cell, pituitary, and mammary tumors. NOR combined with mestranol increased incidences of pituitary, mammary, vaginal, and cervical tumors in female mice and of various other tumors in male mice. NOR was reported to be embryolethal in some species and to have teratogenic effects in mice. Human data are not available on NOR alone. Therefore, it is concluded that there is limited evidence for the carcinogenicity of NOR alone and in combination with mestranol in experimental animals. In humans, NOR is implicated in the effects of oral contraceptives which have been causally related:benign liver adenomas increase and benign breast disease decrease.^ieng

17beta-oestradiol and Enovid mammary tumorigenesis in C3H/HeJ female mice: counteraction by concurrent 2-bromo-alpha-ergocryptine.

Chronic administration of 17beta-oestradiol (via drinking water) or the oral contraceptive Enovid (norethynodrel and mestranol) (0-1 mg injected s.c. twice weekly) to nulliparous C3H/HeJ female mice, beginning at one month of age and terminating at 20 months (17beta-oestradiol) or 22 months (Enovid), significantly increased the incidence of mammary tumours over solvent-treated controls. Concurrent treatment of the steroid-treated mice with 2-bromo-alpha-ergocryptine (CB-154) (0-1 mg s.c. injected daily) significantly reduced mammary tumour incidence and mammary hyperplastic nodule development to the control level. CB-154 is an efficacious inhibitor of pituitary prolactin secretion. These results demonstrate that steroid-induced mammary gland dysplasias can be sharply reduced by chronic CB-154 treatment, and suggest that some of the mammary tumorigenic activities of oestrogenic steroids in C3H mice are mediated via an increased secretion of pituitary prolactin.

Hepatic adenomas and oral contraceptives.

PIP: Over the past 5 years there has been an increase in the number of reports of patients with hepatic adenomas, and an association has been found between these tumors and the use of oral contraceptives. Up to January 1975 46 patients have been reported with this association. The histology of the tumors varies, with several names having been applied to the variations. Adenoma or focal nodular hyperplasia seem to be the most appropriate terms. Most of the commonly used oral contraceptives have been involved. Usually the use of the contraceptive has been at least 2 years. In 1 case, adenoma was diagnosed 4 years after stopping use of the drug. Symptoms have been abdominal pain and an abdominal mass. The tumor may rupture with hemorrhage into the abdominal cavity creating an emergency. Hepatic arteriography has been used to make an early diagnosis. Biochemical tests remain normal. Needle biopsy is contraindicated; surgery is indicated, however. The lesion may be multiple. Hepatic adenoma should be suspected in any young woman with abdominal pain and enlargement of the liver. Ruptured hepatic adenoma should be considered in acute abdominal emergencies, in young women who are taking oral contraceptives, and in older women taking hormone replacements.^ieng

Effects of the antifertility drug enovid in five strains of mice, with particular regard to carcinogenesis.

PIP: The antifertility drug, Enovid, was tested for possible carcinogenicity in female mice of 5 specially selected strains: BALB/c, C3H, C3HfB, A, and C57BI. Enovid was chosen for testing since it is one of the most widely used oral contraceptives. The 5 strains of mice provided maximum genetic variation in the test animals. The drug was fed at 3 dose levels: 5 mcg/gm, 10 mcg/gm and 20 mcg/gm of food. The lowest dose did not prevent reproduction. The 10 mcg dose prevented some females from reproducing. The 20 mcg dose prevented all females from reproducing. The strains of mice differed in their response to Enovid. Weight gain was reduced in all strains. Effect on life-span varied, partly because of the tumors. Cervical and vaginal lesions showed invasion of the epithelium into the stroma but was limited, with few exceptions to the BALB/c females. In the BALB/c strain these lesions occurred in controls as well, but showed more progression and a higher incidence with the highest dose of Enovid. None of these lesions appeared grossly as tumors and none had extended beyond the vaginal wall or metastasized. They were observed only on histologic sections. Neither ovarian nor mammary gland tumors were increased in any strain. In the C3H strain such tumors seemed to be inhibited. In the C3HfB strain there was some inhibition of hepatomas and in the BALB/c strain some inhibition of adrenocortical adenoma. Chromophobe adenomas of the hypophysis were significantly increased in old C57BI females treated with the highest dose of Enovid. Offspring of Enovid-treated females showed no abnormalities. Enovid increased the occurrence and may have advanced the progression of epithelial lesions of the cervix and vagina of old BALB/c females. A study of the lesions in untreated females of this strain might help the understanding of carcinoma in situ in women and possibly the appearance of adeno-carcinoma of the vagina of young women whose mothers had been treated with stilbestrol during the first trimester to maintain pregnancy. Other neoplasms in this strain were not increased by the Enovid therapy. In the C3H strain mammary tumors were reduced by the Enovid and those that did occur were found later than in controls. Results from experimental animals should be applied to humans with care. Such results are of greatest value in directing attention to certain areas for investigation.^ieng