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Lansoprazole increases testosterone metabolism and clearance in male Sprague-Dawley rats: implications for leydig cell carcinogenesis

https://doi.org/10.1016/S0041-008X(03)00275-8Get rights and content

Abstract

Leydig cell tumours (LCTs) are frequently observed during rodent carcinogenicity studies, however, the significance of this effect to humans remains a matter of debate. Many chemicals that produce LCTs also induce hepatic cytochromes P450 (CYPs), but it is unknown whether these two phenomena are causally related. Our aim was to investigate the existence of a liver–testis axis wherein microsomal enzyme inducers enhance testosterone metabolic clearance, resulting in a drop in circulating hormone levels and a consequent hypertrophic response from the hypothalamic–pituitary–testis axis. Lansoprazole was selected as the model compound as it induces hepatic CYPs and produces LCTs in rats. Male Sprague-Dawley rats were dosed with lansoprazole (150 mg/kg/day) or vehicle for 14 days. Lansoprazole treatment produced effects on the liver consistent with an enhanced metabolic capacity, including significant increases in relative liver weights, total microsomal CYP content, individual CYP protein levels, and enhanced CYP-dependent testosterone metabolism in vitro. Following intravenous administration of [14C]testosterone, lansoprazole-treated rats exhibited a significantly smaller area under the curve and significantly higher plasma clearance. Significant reductions in plasma and testicular testosterone levels were observed, confirming the ability of this compound to perturb androgen homeostasis. No significant changes in plasma LH, FSH, or prolactin levels were detected under our experimental conditions. Lansoprazole treatment exerted no marked effects on testicular testosterone metabolism. In summary, lansoprazole treatment induced hepatic CYP-dependent testosterone metabolism in vitro and enhanced plasma clearance of radiolabelled testosterone in vivo. These effects may contribute to depletion of circulating testosterone levels and hence play a role in the mode of LCT induction in lansoprazole-treated rats.

Introduction

Leydig cell hyperplasia and tumours (LCTs) are frequently observed during rodent carcinogenicity studies with a diverse range of chemical agents; however, the significance of this effect to humans remains a matter of debate Cook et al., 1999, Clegg et al., 1997. Mechanistic studies have identified several modes of LCT induction involving interference with the hypothalamic–pituitary–testis (HPT) axis at various points (e.g., androgen receptor antagonists, testosterone biosynthesis inhibitors, 5α-reductase inhibitors, oestrogen agonists, and dopamine agonists) leading to increased pituitary luteinising hormone (LH) secretion (Cook et al., 1999). It is widely accepted that sustained elevation of circulating LH levels can produce Leydig cell hyperplasia and tumours in rodents Christensen and Peacock, 1980, Chatini et al., 1990, Brown et al., 1979.

Many chemicals that produce LCTs also induce hepatic cytochromes P450 (CYPs) (e.g., oxazepam, endosulfan, lansoprazole, and methyl tert-butyl ether (MTBE)), however, it is currently unknown whether these two phenomena are causally related Diwan et al., 1986, Wilson and Le Blanc, 1998, Masubuchi et al., 1997, Williams and Borghoff, 2000. CYPs catalyse key reactions in the biosynthesis and catabolism of steroid hormones, and consequently play a role in steroid hormone homeostasis. Exposure of rodents to microsomal enzyme inducers alters the composition of the hepatic CYP complement, resulting in concomitant changes in biotransformation.

In many instances, enzyme induction of the CYP supergene family is accompanied by toxicity in the target tissue and has been rationalised by induction of specific CYP forms that metabolise pro-carcinogens to biologically reactive intermediates, the latter being responsible for the observed toxicity, including carcinogenesis. The best documented example of this phenomenon is induction of CYP1A1 by polycyclic aromatic hydrocarbons mediated by the Ah receptor, resulting in the formation of mutagenic/carcinogenic diol-epoxides in target tissues, including the liver (Whitlock, 1999). An extension of this phenomenon is that induction of CYPs is well known to modulate the biotransformation of endogenous compounds, including steroids such as testosterone (Waxman, 1988, Sonderfan et al., 1987), resulting in the possible perturbation of cellular homeostasis and predisposition to inducer-dependent toxicity.

We hypothesise that microsomal enzyme inducers produce LCTs in rodents through enhanced metabolic clearance of testosterone (Fig. 1). The resultant decrease in circulating hormone levels would stimulate a compensatory increase in pituitary LH secretion. This concept of the existence of a liver–testis axis is analogous to the liver–thyroid axis, which has an established role in the induction of thyroid hyperplasia and tumours by microsomal enzyme inducers McClain, 1992, McClain et al., 1989.

Four major pathways contribute to the hepatic biotransformation of testosterone to form more polar products that are excreted in the urine or bile. First, testosterone may undergo regio- and stereospecific hydroxylation reactions catalysed by CYP enzymes Waxman, 1988, Sonderfan et al., 1987. As certain of these hydroxylation reactions are catalysed by a single CYP form they can be used as probes to monitor the levels of expression of individual CYP forms (e.g., 2α-OHT for CYP2C11; 7α-OHT for CYP2A1; 6β-/2β-OHT for CYP3A1/2) (Sonderfan et al., 1987). Second, testosterone, or its hydroxylated metabolites, may be conjugated with glucuronic acid or sulfate in reactions catalysed by UDP-glucuronosyltransferase (UDP-GT) and sulfotransferase (ST) enzymes, respectively (Matsui et al., 1974). Finally, testosterone may undergo dehydrogenation to androstenedione catalysed by CYPs and 17β-hydroxysteroid dehydrogenase (17β-HSD) Sonderfan et al., 1987, Wood et al., 1983, Martel et al., 1992. The liver is the major site of androgen inactivation, but the testis has also been demonstrated to catalyse several pathways of testosterone metabolism Sonderfan et al., 1989, Lacroix et al., 1975. Indeed, local formation of androgen metabolites, which might possess unique biological activities, may underlie important physiological roles within the testis.

At present, a limited number of studies have been published regarding the impact of microsomal enzyme inducers on androgen homeostasis in rodents. Wilson and Le Blanc (1998) reported that the organochloride pesticide, endosulfan, induced hepatic CYP-dependent testosterone metabolism and enhanced the urinary elimination of radiolabelled testosterone in female CD-1 mice. However, this was associated with a small, nonsignificant decrease in serum testosterone levels, suggesting that homeostatic feedback mechanisms were able to compensate for the effects of this compound. MTBE is an oxygenated fuel additive, which produces a dose-dependent increase in the incidence of LCTs in rats Bird et al., 1997, Belpoggi et al., 1995. Mechanistic studies have demonstrated induction of CYPs involved in testosterone metabolism (e.g., CYP2B1/2, CYP3A1/2, and CYP2A1), but it is currently unknown whether this underlies the significant reduction in serum testosterone levels observed in MTBE-treated rats Williams and Borghoff, 2000, Williams et al., 2000. A similar mechanism has also been proposed to explain the induction of LCTs by oxazepam and felbamate, although there is currently no direct evidence to support this (Cook et al., 1999).

The aim of the current study was to investigate the existence of a liver–testis axis in rats using lansoprazole as a model compound. Lansoprazole is a gastric proton pump inhibitor and chronic exposure produces Leydig cell hyperplasia and tumours in rats (Atkinson et al., 1990; unpublished report by Takeda Chemical Industries Ltd., A-29-681). Fort et al. (1995) reported that providing testosterone supplementation to lansoprazole-treated rats completely suppressed the induction of LCTs, indicating that reduced negative feedback of testosterone at the hypothalamus and/or pituitary gland might be involved in the mode of LCT induction. Lansoprazole treatment produces a reduction in endogenous testosterone levels, which is primarily attributed to the ability of this compound to inhibit testicular testosterone biosynthesis (Fort et al., 1995). However, induction of hepatic CYP enzymes has been reported in female rats following lansoprazole treatment; therefore, enhanced biotransformation and clearance might contribute to the depletion of circulating testosterone levels (Masubuchi et al., 1997). In this study we have examined the effects of treatment of male rats with lansoprazole on hepatic and testicular CYP expression and resultant changes in hepatic and testicular testosterone metabolism in vitro and testosterone clearance in vivo. We have also investigated the effects of lansoprazole on the endocrine control of the testis.

Section snippets

Animal treatment

Male Sprague-Dawley rats (6- to 8-weeks old; Bantin & Kingman (Hull, UK) (study 1 and 2) or Charles River, UK (study 3)) were housed at 21 ± 1°C, 55 ± 15% humidity and with a 12-h light/dark cycle. Animals had free access to food (Rat and Mouse Expanded Diet (Bantin and Kingman, Hull, UK) (study 1 and 2) or R&M No.1 (Special Diet Services Ltd., UK) (study 3) and water. Following 7 days acclimatisation, rats were dosed once daily with vehicle (0.5% CMC/0.1% Tween 80) or lansoprazole (150

Body and organ weights

Final body weights and relative liver and testes weights of animals from study 1 are shown in Table 1. Final body weights of drug-treated animals were significantly lower compared to the control group. Lansoprazole treatment was associated with significant increases in relative liver and testes weights, although no effect on absolute organ weights was observed (data not shown). Animals were examined daily for clinical signs related to treatment and appeared healthy throughout the dosing period.

Hepatic cytochrome P450 expression

Discussion

Treatment of rats with lansoprazole is associated with reductions in endogenous testosterone levels and an increased incidence of Leydig cell hyperplasia and tumours Fort et al., 1995, Atkinson et al., 1990; unpublished report by Takeda Chemical Industries Ltd., A-29-681). Induction of hepatic CYPs has also been reported in lansoprazole-treated female rats (Masubuchi et al., 1997). We hypothesised that enhanced testosterone metabolic clearance might contribute to the depletion of circulating

Acknowledgements

The authors gratefully acknowledge Peter Kentish, Ruth Storer, and Louise Reid for their technical assistance, Dr. Craig Lambert for assistance with the pharmacokinetic analysis, and Dr. Alex Bell for his scientific input.

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