Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human CYP1B1.

The cytochrome P450 enzymes (P450s or CYPs) are a superfamily of hemeproteins that catalyze the monooxygenation of a wide range of endobiotic and xenobiotic substrates. A typical strategy in toxicological research and testing involves applying a toxicant at high doses for a short period to homogeneous animals under controlled conditions. However, the conditions of this approach have very little in common with actual human exposure. Transgenic (Tg) mice carrying human genes encoding a drug-metabolizing enzyme (CYP) offer a solution to many of the difficulties in the evaluation of chemical toxicity. It has been demonstrated that the expression of human CYP transgenes under the control of mammalian-inducible promoters exhibits relatively poor fold increases after induction. In this study, we used the tetracycline-regulated (tet) promoter system to increase the expression of the human CYP1B1 (hCYP1B1) gene in the tissues of transgenic mice. By mating two lineages of transgenic mice, double transgenic (dTg) mice expressing both tTA and hCYP1B1 genes under the control of the tet promoter were successfully produced, into which the two transgenes were introduced in an embryo. The expression pattern of tTA-driven hCYP1B1 transgene featured a fold induction of more than 3 to 12 in the brain, heart, and lung and 2- to 4-fold induction in the liver, kidney, and intestine upon doxycycline removal. Immunohistochemical staining with hCYP1B1 antibody was also increased by the removal of doxycycline. In addition, the activities of CYP liver microsomes in the dTg mice without doxycycline showed an increase compared to that in the dTg mice treated with doxycycline. The level of activities correspond to the levels of human CYP1B1 protein expression in the Tg mice (-dox) that was increased by 2-fold induction as compared to that of the dTg mice with doxycycline. Thus, overproduction in Tg can be purified and the activity of purified human CYP1B1 can be characterized by alterations to the coding sequence in order to solve the physiological function of this enzyme in a humanized in vivo system. It is also possible to examine the activity of purified human CYP1B1 using several environmental toxicants such as procarcinogens.     

Expression of aquaporin 9 in the adult rat epididymal epithelium is modulated by androgens

Reabsorption of fluid and solutes across the epithelium lining the male excurrent duct is important for adequate sperm maturation, concentration, and storage. Water channels contribute to water movement across epithelia in many tissues. Aquaporin 9 (AQP9) is abundantly expressed in the apical membrane of principal cells that line the epididymis, and in reabsorptive and secretory epithelial cells of the male reproductive tract. In this study we show that the nonsteroidal antiandrogen flutamide, given to adult rats at a dose of 50 mg x kg(-1) x day(-1) for 2 wk via osmotic minipumps significantly decreased the amount of AQP9 in the epididymis. This down-regulation was observed by immunofluorescence of cryostat tissue sections and by Western blotting of epididymal brush border membrane preparations. In addition, castrated adult rats showed lower levels of epididymal AQP9 compared with adult controls, whereas systemic testosterone treatment of castrated adult rats induced a recovery of the expression of AQP9 to control levels. These data indicate that the expression of AQP9, a likely candidate for apical transepithelial fluid and solute transport in several regions of the male reproductive tract, is modulated by androgens in the adult rat epididymis.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

CYP3A4 and pregnane X receptor humanized mice

Marked species differences exist in P450 expression and activities. In order to produce mouse models that can be used to more accurately predict human drug and carcinogen metabolism, P450- and xenobiotic receptor humanized mice are being prepared using bacterial artificial chromosomes (BAC) and P1 phage artificial chromosomes (PAC) genomic clones. In some cases, transgenic mice carrying the human genes are bred with null-mice to produce fully humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 were generated and characterized. Studies with the CYP3A4-humanized (hCYP3A4) mouse line revealed new information on the physiological function of this P450 and its role in drug metabolism in vivo. With this mouse line, CYP3A4, under certain circumstances, was found to alter the serum levels of estrogen resulting in deficient lactation and low pup survival as a result of underdeveloped mammary glands. This hCYP3A4 mouse established the importance of intestinal CYP3A4 in the pharmacokinetics of orally administered drugs. The hCYP3A4 mice were also used to establish the mechanisms of potential gender differences in CYP3A4 expression (adult female > adult male) that could account for human gender differences in drug metabolism and response. The pregnane X receptor (PXR) is also involved in induction of drug metabolism through its target genes including CYP3A4. Since species differences exist in ligand specificity between human and mice, a PXR-humanized mouse (hPXR) was produced that responds to human PXR activators such as rifampicin but does not respond to the rodent activator pregnenalone 16alpha-carbonitrile.     

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Actions of esters of testosterone, dihydrotestosterone, or estradiol on sexual behavior in castrated male guinea pigs

Prepuberally castrated male guinea pigs were treated in adulthood with estradiol benzoate, testosterone propionate, dihydrotestosterone propionate or corn oil (vehicle control). Both corn oil and estradiol benzoate were ineffective in augmenting or inducing any aspect of adult male sexual behavior. Dihydrotestosterone propionate and testosterone propionate were both effective in establishing the complete male sexual behavior pattern, although they differed in the manner in which they affected specific components. For example, males treated with testosterone propionate showed more non-intromissive but not more intromissive mounts than males treated with dihydrotestosterone propionate. In addition, the average frequency of thrusts per intromission was greater for males treated with dihydrotestosterone propionate than for males treated with testosterone propionate.     

Role of endogenous testosterone in myocardial proinflammatory and proapoptotic signaling after acute ischemia-reperfusion

Myocardial ischemia is the leading cause of death in both men and women; however, very little information exists regarding the effect of testosterone on the response of myocardium to acute ischemic injury. We hypothesized that testosterone may exert deleterious effects on myocardial inflammatory cytokine production, p38 MAPK activation, apoptotic signaling, and myocardial functional recovery after acute ischemia-reperfusion (I/R). To study this, isolated, perfused rat hearts (Langendorff) from adult males, castrated males, and males treated with a testosterone receptor blocker (flutamide) were subjected to 25 min of ischemia followed by 40 min of reperfusion. Myocardial contractile function (left ventricular developed pressure, left ventricular end-diastolic pressure, positive and negative first derivative of pressure) was continuously recorded. After reperfusion, hearts were analyzed for expression of tissue TNF-alpha, IL-1beta, and IL-6 (ELISA) and activation of p38 MAPK, caspase-1, caspase-3, caspase-11, and Bcl-2 (Western blot). All indices of postischemic myocardial functional recovery were significantly higher in castrated males or flutamide-treated males compared with untreated males. After I/R, castrated male and flutamide-treated male hearts had decreased TNF-alpha, IL-1beta, and IL-6; decreased activated p38 MAPK; decreased caspase-1, caspase-3, and caspase-11; and increased Bcl-2 expression compared with untreated males. These results show that blocking the testosterone receptor (flutamide) or depleting testosterone (castration) in normal males improves myocardial function after I/R. These effects may be attributed to the proinflammatory and/or the proapoptotic properties of endogenous testosterone. Further understanding may allow therapeutic manipulation of sex hormone signaling mechanisms in the treatment of acute I/R.     

Bisphenol S induces oxidative stress-mediated impairment of testosterone synthesis by inhibiting the Nrf2/HO-1 signaling pathway

Bisphenol S (BPS) is an environmental endocrine disruptor widely used in industrial production. BPS induces oxidative stress and exhibits male reproductive toxicity in mice, but the mechanisms by which BPS impairs steroid hormone synthesis are not fully understood. Nuclear factor erythroid 2-related factor 2(Nrf2)/HO-1 signaling is a key pathway in improving cellular antioxidant defense capacities. Therefore, this study explored the effects of exposure to BPS on testosterone synthesis in adult male mice and its mechanisms with regard to the Nrf2/HO-1 signaling pathway. Adult male C57BL/6 mice were orally exposed to BPS (2, 20, and 200 mg/kg BW) with sesame oil as a vehicle (0.1 ml/10 g BW) per day for 28 consecutive days. The results showed that compared with the control group, serum testosterone levels were substantially reduced in the 20 and 200 mg/kg BPS treatment groups, and testicular testosterone levels were reduced in all BPS treatment groups. These changes were accompanied by a prominent decrease in the expression levels of testosterone synthesis-related enzymes (STAR, CYP11A1, CYP17A1, HSD3B1, and HSD17B3) in the mouse testis. In addition, BPS induced oxidative stress in the testis by upregulating the messenger RNA and protein levels of Keap1 and downregulating the levels of Nrf2, HO-1, and downstream antioxidant enzymes (CAT, SOD1, and Gpx4). In summary, our results indicate that exposure of adult male mice to BPS can inhibit Nrf2/HO-1 signaling and antioxidant enzyme activity, which induces oxidative stress and thereby may impair testosterone synthesis in testicular tissues, leading to reproductive damage.     

Female-typical sexual behavior of rhesus and defeminization by androgens given prenatally

Adult rhesus monkeys were observed in standardized tests for female-typical sexual and related social responses. In the first experiment reported, 7 castrated males and 5 spayed females were paired with each of 4 intact males on two occasions following intramuscular injection with estradiol benzoate (EB) (6 micrograms/kg X 14 days) and on two other occasions without such treatment. In tests without EB, males and females did not behave differently toward the intact male partners, and all responses were displayed at low frequencies. In tests with EB, females showed reliably higher frequencies than males of approaching, sitting close to, grooming, and soliciting, and they presented to a higher proportion of the male partner's sexual contacts. EB reliably increased the frequency of display of all of these same five responses in females but not in castrated males. The intact male partners displayed reliably fewer approaches, sexual contacts, mounts, intromissions, and ejaculations to castrated males than to spayed females regardless of estrogenization. In a second experiment 10 intact adult pseudohermaphroditic females and 6 intact control females were tested following EB injections with each of the same 4 intact males. Pseudohermaphrodites were experimentally produced by injecting pregnant females with either testosterone propionate (TP) or dihydrotestosterone propionate (DHTP). Pseudohermaphrodites, regardless of type of androgen used in their production, showed reliably fewer solicits than controls to male partners. Moreover, they displayed most of the other responses at lower average frequencies than controls. Frequencies of intromission and ejaculation by intact male partners were reliably lower with pseudohermaphrodites than with control females, but frequencies of approach, sexual contact, and mount were not reliably different. We conclude that in this testing and measurement situation male and female rhesus monkeys differ markedly in the degree of expression of female-typical sexual behaviors, and genotypic males are behaviorally less responsive to estrogens than females. Exposing genotypic females to androgens during fetal life decreases the expression of female-typical, estrogen-influenced responses, and the effect is most pronounced on those soliciting responses that subserve proceptivity.     

Androgen influence on male mouse ultrasounds during courtship

Ultrasonic vocalizations are emitted by adult male mice (Mus musculus) shortly after an adult female or her odor are encountered, possibly as an early part of courtship behavior. Consistent with this hypothesis, it was found that castration of adult male mice increased the latency to first ultrasonic vocalization in response to an adult female. In addition, castrated males, subsequently injected once with 200 μg of testosterone propionate, reduced their latency, whereas oil-injected castrates did not.     

Role of postnatal androgens in sexual differentiation of the lordosis-inhibiting effect of central injections of cholecystokinin

The neuropeptide cholecystokinin (CCK) inhibits lordosis behavior when infused into the ventromedial nucleus of the hypothalamus (VMN) of female rats and has no effect when infused into the VMN of male rats. To test whether this sex difference develops under the control of perinatal steroids, male rats were castrated or given sham surgeries within 3 h of birth and female rats were injected with either 0 or 100 micrograms testosterone propionate on postnatal day 5. As adults, these rats were castrated as necessary, implanted with unilateral cannulae directed at the VMN, and tested for their ability to display female sexual behavior and to respond to CCK. Neonatal castration of males prevented defeminization of this response. When treated with 5 micrograms estradiol benzoate (EB), neonatally castrated males showed both lordosis behavior and a profound inhibition of that behavior after infusions of CCK. Neonatally castrated males did not display lordosis behavior when treated with 2 micrograms EB. Control males showed no lordosis behavior and, therefore, no response to CCK. Both doses of EB induced lordosis behavior in neonatally androgenized females. Significantly, these neonatally androgenized females were less responsive to CCK's inhibition of lordosis and were also anovulatory. These results imply that androgens alter the development of CCK responsive circuits as well as defeminize cyclic gonadotropin release. Levels of 125I-sCCK-8 binding in the VMN were correlated closely with an individual's ability to respond to sCCK-8. In summary, the inhibition of female sexual behavior caused by exogenously administered CCK in normal adult female rats appears to be controlled at least partially by levels of CCK receptors in the VMN and to differentiate under the control of perinatally present testosterone.     

Regulation of the expression of protein kinase C isoenzymes in rat ventral prostate: effects of age,castration and flutamide treatment

Protein kinase C (PKC) isoenzymes are involved in cell function, growth, apoptosis and neoplastic transformation in the prostate gland. We detected by means of Western blot the expression of the classical alpha and beta1, the novel epsilon and the atypical zeta isoforms of PKC in ventral prostates from rats with different extents of plasma testosterone levels and/or androgen imprinting on the gland. The expression of the four isoforms decreased in 5-day castrated rats showing apoptotical regression of the gland and a drastic reduction of circulating testosterone. However, the expression of PKC isoenzymes (alpha, beta1, epsilon ) increased in prostates from pubertal (35-days old) rats that are characterized by relatively low but extremely bioactive testosterone plasma levels. Treatment of adult rats for 14 days with flutamide (daily s.c. injection of 15 mg/Kg B.W.) resulted in increased expression of the four isoenzymes; it occurred in the presence of increased (normal rats) or drastically reduced (rats castrated after 9 days of flutamide administration) levels of plasma testosterone conceivably through a direct effect of this nonsteroidal antiandrogen on prostate cells. Measurements of PKC(alpha) activity were in agreement with the observations on protein expression and showed that flutamide (that is extensively used in the treatment of advanced prostate cancer) elicits some impairment in the mechanisms of translocation of this isoform from the cytosol to the membrane. Thus, in addition to the possibility of direct effects of flutamide upon the rat prostate, we present evidence that the levels of circulating androgens and/or their bioactivity in the gland regulate the expression of various important PKC isoforms.     

Sexual preferences of male hamsters (Mesocricetus auratus) for conspecifics in different endocrine conditions

The behavioral responses of sexually experienced male hamsters toward a pair of anesthetized conspecifics were investigated. Males spent significantly more time licking, sniffing, and mounting neonatally and adult castrated males than intact males. Adult castrated males receiving oil injections were preferred over castrates receiving exogenous testosterone propionate (TP). Ovariectomized females were preferred over intact males, adult castrated males, or spayed females receiving exogenous TP. It was concluded that the absence of an androgen-dependent factor(s) renders an animal more sexually attractive.     

Hormone effects on male sex behavior in adult South African clawed frogs, Xenopus laevis

Intact, adult male Xenopus laevis were injected with human chorionic gonadotropin (HCG) and tested with intact HCG-primed females. Under these conditions, males displayed high levels of sex behavior (clasping of females). By 2 weeks after castration, these males had ceased clasping. Testosterone and dihydrotestosterone reinstated clasping in male castrates. Following removal of testosterone or dihydrotestosterone pellets, castrated males ceased to clasp. No male was ever observed to clasp following estradiol implanted in pellets or in silastic capsules. In experiments on castrated, adult, female Xenopus laevis, both testosterone and testosterone propionate pellets reliably produced male sex behavior in the form of clasping. The clasping of testosterone-implanted female and male castrates was very similar in form and duration. The behavioral effectiveness of testosterone in both sexes and the ineffectiveness of estradiol in eliciting clasping is paralleled by autoradiographic localization of sex steroids in brain where the distribution of testosterone-concentrating, cells is the same for males and females, but different from the distribution of estradiol-concentrating cells.     

Androgenic stimulation of aggression eliciting cues in adult opponent mice castrated at birth, weaning, or maturity

The developmental and concurrent effects of androgens on aggression-eliciting qualities of male opponent mice were investigated during paired contests with trained fighters. Groups of male mice were castrated at either 1, 20, or 110 days of age. Half of each group were injected from 110 days of age with a maintenance dose of 100 αg testosterone propionate (TP), while the rest received injections of the arachis oil vehicle. The level of aggression received from fighter mice was monitored after 20 injection days, and compared to that received by intact male, and spayed TP-injected female opponents. The age at castration did not affect the responsiveness of opponents to androgens, and all TP-injected males suffered more severe defeat than oil-injected controls. TP-injected castrate males did not differ from intact males as opponents eliciting aggression, though TP-treated females received significantly more bites than any of the male opponent groups. A concurrent stimulation by androgens of the adult males' aggression-eliciting cues was, therefore, demonstrated. It is suggested that females derive different metabolic end-products from testosterone propionate, which can apparently provide more effective aggression-eliciting cues than are produced by the male mouse.     

Testosterone-induced aggression in adult female mice

Silastic implants of testosterone (T) and injections of testosterone propionate (TP) were used to study the effects of ovariectomy and androgen administration on the fighting behavior of adult female mice. A dose of T previously shown to hyperstimulate accessory organ growth in adult male castrates was sufficient to induce the complete behavioral repertoire of male-like aggression in females never before treated with exogenous androgen. As determined by radioimmunoassay, blood levels of T produced by implants containing an aggression-inducing dose of T (10-mg implant) were within the range of T concentrations observed in intact males. Following treatment with a 10-mg T implant, the aggressive behavior of ovariectomized females could be fully maintained with a dose of T (0.3-mg implant) that failed to maintain weight of the accessory organs in adult male castrates. In fact, females “androgenized” were subsequently more responsive to the aggression-activating properties of T than were males castrated after prenatal and perinatal androgen exposure.     

Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice

Little is known about the hormonal regulation of sexual behavior and about the pattern of expression in the brain of sex-steroid receptors in the BALB/c AnN strain of mice (Mus musculus). In this study, 8-week old male BALB/c AnN mice were castrated and the temporal course of decline of sexual behavior was studied, as well as the effects of daily treatment with either testosterone propionate (TP), estradiol benzoate (EB), or dihydrotestosterone propionate (PDHT). Castration resulted in rapid decline of sexual behavior, in both control or vehicle-treated mice. TP maintained full sexual behavior of castrated mice, while PDHT or EB did not have this effect. The expression of ER-alpha dropped nearly 50% after castration, and this pattern remained in TP or PDHT-treated mice, while EB increased the ER-alpha mRNA levels to almost the same values as in intact control mice. The same pattern was found when ER-beta mRNA levels were analyzed. The expression of the PR-A/B gene in the different brain regions in intact mice and after castration, or among the differently treated mice, showed significant differences between normal and castrated mice at all times in all brain regions studied, with the exception of the frontal cortex. Castration reduced the expression of AR by 10-fold, as compared to intact control mice, while TP or PDHT treatment returned its expression to the same levels as in intact control mice, in all brain areas studied. The changes are more prominent in POA-HIP than in HYP and CF. These results demonstrated a rapid decline of sexual behavior in this strain of mice after castration, and show that only TP was able to maintain male sexual behavior, with no correlation with the pattern of expression of sex hormone receptors in specific areas of the mouse brain.     

Parental responsiveness is feminized after neonatal castration in virgin male prairie voles, but is not masculinized by perinatal testosterone in virgin females

We previously found a large sex difference in the parental responsiveness of adult virgin prairie voles (Microtus ochrogaster) such that most males are spontaneously parental, whereas most females are not. Because this sex difference is independent of the gonadal hormones normally circulating in adult virgin voles, the present study examined whether perinatal hormones influence the development of this sex difference. Males were treated prenatally (via their pregnant dam) with both the androgen receptor blocker flutamide (5 mg/day/dam) and the aromatase inhibitor ATD (1 mg/day/dam), or oil, for the last 2 weeks of gestation. Half of the subjects from each group were castrated on the day of birth and the other half received a sham surgery. As adults, intact males were castrated and all males received a silastic capsule filled with testosterone. Prenatal treatment with flutamide and ATD had no effect on males' behavior toward pups, but neonatal castration significantly reduced the percentage of males acting parentally. In a second experiment, females were exposed to testosterone propionate (TP; 50 microg/day/dam) or oil via their dam during the last 2 weeks of gestation. For the first neonatal week, half of the females from each group were injected with TP (1 mg/day) and the other half oil. As adults, females were ovariectomized and half from each group received a testosterone-filled capsule and the other half received an empty capsule. None of the perinatal TP treatments increased females' parental responsiveness, although females from all groups that received testosterone capsules as adults were highly parental. Therefore, although postnatal testicular hormones are necessary for high parental responsiveness in males, the behavior of females is not influenced by perinatal exposure to testosterone.     

Effect of sex hormones on the 1,2-dimethylhydrazine metabolic activity in the kidneys of CBA-strain mice

1,2-dimethylhydrazine (DMH) metabolizing activity of kidney microsomes was shown to be two times higher in male, than in female CBA mice. Castration decreased DMH metabolizing activity of male kidney microsomes to the females' level. DMH metabolizing activity of castrated males treated with testosterone propionate was identical to that of intact males. The incorporation of 14C-DMH into kidney DNA was also higher in male, than in female CBA mice.