Profound effects of burn and ethanol on proinflammatory cytokines of the reproductive axis in the male mouse.

Thermal injury is often associated with previous ethanol exposure, and close to 50% of patients admitted to a burn unit have a potentially high blood ethanol level. Cellular mechanisms by which ethanol and/or burn affect the hypothalamic-pituitary-gonadal (HPG) axis are not entirely understood. However, it is known that the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 influence negatively on the endocrine functions of the HPG. We report a time course study (6, 12, 24, and 48 hours) of the effects of ethanol, burn, or the combination of burn/ethanol on proinflammatory cytokines of the hypothalamus, pituitary and testes of male C57Bl/6 mice. We found that there were highly significant increases in each of these cytokines caused by ethanol, burn, and burn/ethanol compared with sham/vehicle (P < .001). This was true in hypothalamus, pituitary, and testes. Because these cytokines generally reduce reproductive function, it may be that proinflammatory cytokines of HPG axis mediate the deleterious effects of burn and/or ethanol on mammalian reproduction.

Vitamin E prevents ethanol-induced inflammatory, hormonal, and cytotoxic changes in reproductive tissues.

Ethanol causes decreased function of the hypothalamic-pituitary-gonadal (HPG) axis. Ethanol resulted in inflammatory changes in HPG manifested by increased concentrations of pro-inflammatory cytokines. Since, such cytokines have deleterious effects on functions of HPG, it seemed possible that ethanol's suppressive action could be due, at least in part, to this inflammation. Since oxidative stress can cause inflammation, we have used the antioxidant vitamin E to test, whether reducing inflammation might protect reproductive functions from ethanol. Rats were fed an ethanol diet or pair fed identically without ethanol for a 3-week period. For the last 10 days, animals were given 30 IU/kg or 90 IU/kg or vehicle. Ethanol significantly increased hypothalamic, pituitary and testicular TNF-alpha and IL-6, all changes prevented by the higher dose of vitamin E. Also, ethanol induced changes in LHRH, LH, testosterone, and testicular germ cell apoptosis were similarly prevented by vitamin E. These data strikingly show that vitamin E protects the HPG from deleterious effects of ethanol and suggests that the mechanism of this protection might be both anti-inflammatory and antioxidant.

Effects of insulin on hepatic inflammation induced by ethanol and burn injury in a murine model of critical illness.

In recent, landmark clinical trials, insulin to maintain euglycemia in critically ill patients improved clinical outcomes, including decreased all-cause mortality. Novel antiinflammatory effects of insulin have recently been described. Thermal injury is an excellent model of critical illness. The addition of ethanol to the model is of great clinical relevance because nearly 50% of the patients admitted to hospitals for burn injuries have ethanol in their circulation. Utilizing a murine model of critical illness (ethanol and skin burn), we tested the hypothesis that insulin treatment in ethanol-exposed, burn-injured mice reduced hepatic inflammation, a potential mechanism for the benefit of insulin. Adult male C57BL/6 mice were given a single intraperitoneal injection of ethanol or saline, were given a 15% total body full-thickness skin burn, or were sham-burned and killed 24 hours later. In each group, half the animals were given subcutaneous injections of the long-lasting basal insulin glargine; the other half, the appropriate vehicle. Hepatic inflammatory markers, including polymorphonuclear infiltration, a chemokine, an important adhesion molecule, proinflammatory cytokines, and nuclear factor kappaB, were measured, and all were increased by ethanol and/or burn. These increases were prevented by insulin. An antiinflammatory cytokine was reduced by ethanol and/or burn. Insulin prevented this decrease. Thus, insulin has a substantial antiinflammatory effect, and this may underlie its dramatic clinical benefit in critical illness.

Effects of chronic ethanol (EtOH) administration on pro-inflammatory cytokines of the hypothalamic-pituitary-gonadal (HPG) axis in female rats.

We and others have investigated the effects of acute and chronic ethanol (EtOH) administration on function of the hypothalamic-pituitary-gonadal (HPG) axis in female rats, consistently finding EtOH to be detrimental. There are now substantial data that pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFalpha) and interleukin 6 (IL-6), have anti-reproductive effects. If EtOH increased levels of these cytokines, such data would be consistent with, though not necessarily prove, a cytokine mediated mechanism for EtOH's deleterious effects on reproduction. Young adult female Sprague Dawley rats were used. In the experiment reported here, the Lieber DeCarli diet was used, with animals fed a 36% EtOH containing diet or pair fed an identical diet which contained dextrimaltose instead of EtOH. This was done for 4 to 6 weeks. TNFalpha and IL-6 were measured in the hypothalamus, pituitary, and ovary by ELISA. EtOH exposure resulted in significant increases in TNFalpha and IL-6 in hypothalami, pituitaries, and ovaries. The data reported here are the first to show consistent stimulatory effects of EtOH exposure on cytokines in the reproductive axis of female rats. Because the effects of these cytokines are generally anti-reproductive, these data provide a rational for more rigorous testing of the notion that part of EtOH's deleterious HPG effects may be due to such immuno-endocrine interactions.

Ethanol-induced alterations in Rab proteins: possible implications for pituitary dysfunction.

Chronic exposure of pubertal male rats to ethanol results in a decline in serum testosterone, increased gonadotropins, pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) content, and decreased or inappropriately normal serum LH and FSH levels, suggesting impaired secretory release of gonadotropins. The molecular mechanisms behind this disorder are undefined, but a disruption of vesicle-mediated secretory processes is possible because intracellular protein trafficking pathways are involved in secretion of glycoproteins such as FSH and LH. Because small GTP-binding proteins of Rab family have been implicated as key regulators of membrane and protein trafficking in mammalian cells, this study was designed to test if ethanol-impaired pituitary FSH and LH secretion is associated with changes in Rab proteins, particularly Rab1B, Rab3B, Rab6, and Rab11. Male Sprague-Dawley rats 35 days old were pair-fed a Lieber-DeCarli diet with ethanol or without ethanol for 5 to 60 days. After ethanol exposure, serum testosterone levels decreased while LH and FSH were inappropriately unchanged. Immunohistochemical staining showed decreased Rab1B, Rab3B, and Rab11 protein levels in ethanol-treated pituitaries. Immunoblotting showed that ethanol induced a transient reduction in Rab6 after 5 days of ethanol exposure, whereas Rab3B decreased after 20 days, Rab11 after 30 days, and Rab1B after 60 days. Despite these changes in Rab proteins, mRNA levels were unaffected by ethanol exposure. We concluded that reductions in key Rab proteins may lead to altered vesicle trafficking and may play a role in disruption of pituitary FSH and LH secretion caused by ethanol.

Exposure to ethanol induces oxidative damage in the pituitary gland.

Chronic exposure of pubertal male rats to ethanol results in a decline in serum testosterone and decreased or inappropriately normal serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels suggesting a functional defect in the pituitary. The molecular mechanisms behind this disorder are undefined. A role for ethanol-induced oxidative damage in the pathophysiology is supported by studies in liver, muscle, and heart of experimental animals, but there is limited evidence in the pituitary. We examined markers of oxidative damage to lipids and proteins in pituitaries from rats consuming ethanol for 5, 10, 20, 30, and 60 days in addition to markers of damage to nucleic acids in pituitaries after 60 days of ethanol exposure. There were increases in 8-oxo-deoxyguanosine immunoreactivity, a marker of oxidative damage to nucleic acids, and an overall increase in malondialdehyde and 4-hydroxynonenal, markers of lipid peroxidation. Protein carbonylation and protein nitrotyrosination, markers of protein oxidation, were significantly increased after 30 days and 60 days of ethanol consumption, respectively. After 60 days of ethanol exposure, TUNEL assay revealed that cell death in the ethanol-treated pituitaries was not significantly different from that in the pair-fed controls at the time of examination. We also measured serum testosterone, FSH, and LH after ethanol consumption for 5, 10, 20, 30, and 60 days. Through 5 to 60 days of ethanol exposure, testosterone levels were consistently lower whereas LH and FSH were inappropriately unchanged, suggesting pituitary malfunction. These results provide evidence for ethanol-induced oxidative damage at the pituitary level, which may contribute to pituitary dysfunction.

The impact of burn injury and ethanol on the cytokine network of the mouse hypothalamus: reproductive implications.

Nearly 50% of the patients admitted to hospitals for burn injuries have detectable levels of alcohol (EtOH) in their circulation. In fact, EtOH is often a causal factor in their injury. It is well known that EtOH as well as burn injury disrupt function of the hypothalamic-pituitary-gonadal (HPG) axis. The cellular mechanisms by which EtOH and/or burn impacts on the HPG are not entirely understood. In the studies reported here, we tested the hypothesis that these injuries mediated their effects by local hypothalamic inflammation. Young adult male mice were subjected to either a 15% total body surface area, full thickness scald, to EtOH, or to both and compared to appropriate controls. They were sacrificed 48 h later. EtOH and burn, as well as the combined injury, consistently and impressively reduced serum testosterone, while increasing hypothalamic concentrations of all three of the pro-inflammatory cytokines, TNFalpha, IL-1beta, and IL-6. In general, the increases induced by burn were greater than those caused by EtOH and the effect of the combined insult was not additive. Hypothalamic concentrations of LHRH were also increased. The data are consistent with the idea that EtOH and/or burn, as models of critical illness, medicate their hypothalamic suppressive effects via increase in pro-inflammatory cytokines.

EtOH disrupts female mammalian puberty: age and opiate dependence.

The major drug of abuse among teenagers in the United States continues to be ethanol (EtOH), but use is seen in children as young as nine. In the studies reported here, the impact of EtOH on biologic and hormonal parameters of puberty was assessed in female rats. Rats were fed a liquid diet containing EtOH, pair fed an identical liquid diet containing dextrimaltose instead of EtOH, or fed a liquid diet not containing EtOH ad libitum. Feeding was started at 21, 25, or 28 d of age. EtOH markedly delayed the age at vaginal opening (34.5 +/- 0.5 d in controls vs 48.5 +/- 2.4 d in EtOH animals; p < 0.001), delayed the age at first estrous (40.9 +/- 0.6 d in controls vs 61.2 +/- 2.6 d in EtOH animals; p < 0.001), increased the length of the estrous cycle, and decreased the number of proestrous days. EtOH, concomitant with reduced ovarian and uterine weight, decreased serum estradiol and progesterone. Associated with these changes in ovarian hormones there was a selective increase in follicle-stimulating hormone, but not luteinizing hormone. EtOH consistently reduced insulin-like growth factor-1. In general, EtOH-induced disruption was more severe the younger the animals were at the start of feeding. Opiate receptor blockade with naltrexone completely prevented the EtOH-induced delay in vaginal opening. The impact of EtOH on female puberty is dramatic, is an emerging public health problem, and deserves more study.