Widely varied chemicals--including certain herbicides, plasticizers, drugs, and natural products--induce peroxisome proliferation in rodent liver and other tissues. This phenomenon is characterized by increases in the volume density and fatty acid oxidation of these organelles, which contain hydrogen peroxide and fatty acid oxidation systems important in lipid metabolism. Research showing that some peroxisome proliferating chemicals are nongenotoxic animal carcinogens stimulated interest in developing mode of action (MOA) information to understand and explain the human relevance of animal tumors associated with these chemicals. Studies have demonstrated that a nuclear hormone receptor implicated in energy homeostasis, designated peroxisome proliferator-activated receptor alpha (PPARalpha), is an obligatory factor in peroxisome proliferation in rodent hepatocytes. This report provides an in-depth analysis of the state of the science on several topics critical to evaluating the relationship between the MOA for PPARalpha agonists and the human relevance of related animal tumors. Topics include a review of existing tumor bioassay data, data from animal and human sources relating to the MOA for PPARalpha agonists in several different tissues, and case studies on the potential human relevance of the animal MOA data. The summary of existing bioassay data discloses substantial species differences in response to peroxisome proliferators in vivo, with rodents more responsive than primates. Among the rat and mouse strains tested, both males and females develop tumors in response to exposure to a wide range of chemicals including DEHP and other phthalates, chlorinated paraffins, chlorinated solvents such as trichloroethylene and perchloroethylene, and certain pesticides and hypolipidemic pharmaceuticals. MOA data from three different rodent tissues--rat and mouse liver, rat pancreas, and rat testis--lead to several different postulated MOAs, some beginning with PPARalpha activation as a causal first step. For example, studies in rodent liver identified seven "key events," including three "causal events"--activation of PPARalpha, perturbation of cell proliferation and apoptosis, and selective clonal expansion--and a series of associative events involving peroxisome proliferation, hepatocyte oxidative stress, and Kupffer-cell-mediated events. Similar in-depth analysis for rat Leydig-cell tumors (LCTs) posits one MOA that begins with PPARalpha activation in the liver, but two possible pathways, one secondary to liver induction and the other direct inhibition of testicular testosterone biosynthesis. For this tumor, both proposed pathways involve changes in the metabolism and quantity of related hormones and hormone precursors. Key events in the postulated MOA for the third tumor type, pancreatic acinar-cell tumors (PACTs) in rats, also begin with PPARalpha activation in the liver, followed by changes in bile synthesis and composition. Using the new human relevance framework (HRF) (see companion article), case studies involving PPARalpha-related tumors in each of these three tissues produced a range of outcomes, depending partly on the quality and quantity of MOA data available from laboratory animals and related information from human data sources.

PPARα Agonist-Induced Rodent Tumors: Modes of Action and Human Relevance

Endocrine regulation of male fertility by the skeleton

Mechanisms of Extrahepatic Tumor Induction by Peroxisome Proliferators in Male CD Rats

I. Introduction II. Neurohormones/Neuropeptides A. GH-releasing hormone (GHRH) B. Pituitary adenylate cyclase-activating peptide (PACAP) C. GnRH D. CRH E. Oxytocin (OT) F. Arginine vasopressin (AVP) G. TRH H. Somatostatin (SRIF) I. Opioids J. Substance-P (SP) K. Neuropeptide Y (NPY) III. Peptides Originally Identified in the Male Gonad A. Inhibin (INH) and activin (ACT) B. PModS IV. Growth Factors A. Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBs) B. Transforming growth factor-β (TGFβ) C. TGFα/EGF D. Fibroblast growth factor (FGF) E. Platelet-derived growth factor (PDGF) F. Nerve growth factor (NGF) G. Steel factor (SLF) H. Gastrin-releasing peptide (GRP) V. Immune Derived Cytokines A. Interleukins (ILs) B. Tumor necrosis factor-α (TNFα) VI. Vasoactive Peptides A. Endothelin (ET) B. Angiotensin-II (AT-II) C. Atrial natriuretic peptide (ANP) VII. Conclusions and Perspectives

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Gonadal Peptides as Mediators of Development and Functional Control of the Testis: An Integrated System with Hormones and Local Environment

Masculinization depends on adequate production of testosterone by the fetal testis within a specific "masculinization programming window." Disorders resulting from subtle deficiencies in this process are common in humans, and environmental exposures/lifestyle could contribute causally because common therapeutic and environmental compounds can affect steroidogenesis. This evidence derives mainly from rodent studies, but because there are major species differences in regulation of steroidogenesis in the fetal testis, this may not always be a guide to potential effects in the human. In addition to direct study of the effects of compounds on steroidogenesis, information also derives from study of masculinization disorders that result from mutations in genes in pathways regulating steroidogenesis. This review addresses this issue by critically reviewing the comparative timing of production and regulation of steroidogenesis in the fetal testis of humans and of rodents and its susceptibility to disruption; where there is limited information for the fetus, evidence from effects on steroidogenesis in the adult testis is considered. There are a number of fundamental regulatory differences between the human and rodent fetal testis, most notably in the importance of paracrine vs. endocrine drives during masculinization such that inactivating LH receptor mutations block masculinization in humans but not in rodents. Other large differences involve the steroidogenic response to estrogens and GnRH analogs and possibly phthalates, whereas for other compounds there may be differences in sensitivity to disruption (ketoconazole). This comparison identifies steroidogenic targets that are either vulnerable (mitochondrial cholesterol transport, CYP11A, CYP17) or not (cholesterol uptake) to chemical interference.

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Steroidogenesis in the Fetal Testis and Its Susceptibility to Disruption by Exogenous Compounds

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells

Catecholamine stimulation of testosterone production via cyclic AMP in mouse leydig cells in monolayer culture

The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

Previous work by Janszen et a1.l indicated that there are two types of Leydig cells in adult rat testes. Recently Schumacher et al.' described the application of the density gradient medium, Percoll, to the purification of mouse Leydig cells. Using this method, the density distribution and activity of mouse and rat Leydig cells have now been investigated. In addition, the functional activity of mouse Leydip cells in monolayer culture has been determined. Crude testis suspensions were centrifuged through continuous gradients (0-90%) of Percoll. Three bands of nucleated cells were obtained with densities of 1.046, 1.053, and 1.070 g/ml. In a typical experiment with mouse Leydig cells, these bands contained 32%, 23%, and 90% Leydig cells; produced 0, 0.08, and 1.35 pg testosterone/lOG Leydig cellsl2 h after stimulation with LH; and bound 21.7, 4.26, and 69.4 fmols [1251]hCG/10G Leydig cells. By subfractionation of the gradients it was demonstrated that LH had little or no effect on testosterone synthesis in Leydig cells of density greater than 1.08 g/ml and less than 1.06 g/ml. Leydig cells of density 1.06-1.08 g/ml produced 1-4 pg testosterone/lO\" Leydig cells/2 h after addition of LH. Similar results were obtained with rat testis cells. Fresh suspensions of mouse Leydig cells of average density 1.070 g/ml were cultured in monolayers, in medium containing 10% fetal calf serum. They retained their original morphology for 24 h after which they gradually spread out to give a typical epithelioid appearance. The LH-stimulated testosterone production of these cells was constant for 2-3 days and then declined. The LH-stimulated cyclic AMP production increased after the first day and then declined by day 3-4. LH receptor levels were maintained for two days and then declined. To conclude. both mouse and rat Leydig cells have been shown to be heterogeneous with respect to their density and steroidogenesis. For the first time it has been demonstrated that adult Leydig cells can be maintained in monolayer culture for 2-3 days without loss in functional activity.

Morag G. Hunter

Papers

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells

Catecholamine stimulation of testosterone production via cyclic AMP in mouse leydig cells in monolayer culture

The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

The heterogeneity and monolayer culture of leydig cells from adult mouse and rat testes