Effect of Nitrous Oxide on Intracellular Events: DISCUSSION

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19 Dec 2009

Effect of Nitrous Oxide

Epidemiologic data from human populations and animal studies indicate that chronic exposure to N20 may result in impaired reproductive function and decreased fertility rates. The mechanism by which N20 might elicit reproductive toxicity is unclear. N20 may interfere with reproductive function at the level of the hypothalamus, pituitary, or reproductive tract. Our previous studies indicate that N20 may have a central nervous system site of action for its antigonadal action. However, the effects of chronic exposure to N20 on the central neu rochemical control of gonadotropin release are at present poorly understood despite the wide use of this agent in dental practice. In this study, we report for the first time direct effects of N20 on GnRH-secreting immortalized hypothalamic neurons.

Exposure to 60% N20 over a 24-hour period resulted in an 80% decrease in GnRH mRNA levels compared with control cells. Exposure to N20 did not affect the viability of GT1-7 cells, indicating that the results observed in this study are not due to toxic effects of the gas on the cells. A decrease in GnRH mRNA levels may arise from a decrease in either the synthesis or stability (increased breakdown) of mRNA. Because GnRH mRNA has a reported half-life of 22-31 hours, it is possible that both a decrease in synthesis of GnRH mRNA and an increased rate of catabolism of mRNA may be involved in order to bring about an 80% decrease in GnRH mRNA levels in N20-exposed cells. Recent studies indicate that elevated intracellular calcium levels inhibit GnRH gene expression as well as the stability of GnRH mRNA.23 Whether N20 interferes with signal transduction elements such as calcium inside the cells remains to be investigated. Furthermore, N20 has also been shown to irreversibly oxidize vitamin B12, rendering it inactive as a coenzyme for certain biochemical reactions, thus resulting in decreased conversion of deoxyuridine and homocysteine to thymidine and methionine, respectively. These effects of N20 are postulated to disrupt DNA synthesis and cause teratogenic effects. Whether N20 disrupts these biochemical events in GT1-7 cells remains to be investigated. It is likely that N20’s ability to decrease GnRH mRNA levels in GT1-7 cells may be secondary to disruption of biochemical processes within the cell. suhagra 100

Exposure to N20 over a period of 24 hours resulted in decreased release of GnRH in response to KC1 stimulation, even though intracellular stores of GnRH were not affected. Because 24-hour exposure to N20 also significantly depressed the GnRH mRNA levels in the GT1-7 cells, it is possible that a reduction in message levels may not immediately translate into decreased GnRH levels at 24 hours of N20 exposure. The cells may have large stores of pro-GnRH, which are processed into the mature GnRH form over 24 hours of N20 exposure. The specific effects of N20 on transna-tional and posttransnational events associated with GnRH production in GT1-7 cells are unknown at present. Given the fact that N20-exposed cells and air-exposed cells had similar intracellular levels of GnRH, the ability of KC1 to stimulate a twofold greater release of the peptide from control cells compared with N2Oex-posed cells is intriguing. In intact rats, studies performed in our laboratory indicate that subchronic N20 exposure results in a disruption of estrous cyclicity, with concomitant alterations in the levels of diencephalic opioid peptides and GnRH. In intact female rats, exposure to N20 on the morning of proestrus results in a threefold increase in total GnRH cell counts in the hypothalamus.

Exposure to N20 also resulted in increased accumulation of GnRH in the preoptic region of rats. Taken together, these results suggest an impaired ability of GnRH cells to release GnRH as a result of N20 exposure. N20 exposure may interfere with evoked release of GnRH from these cells. In addition, N20 may also inhibit synthesis of GnRH mRNA by directly acting on the GnRH neurons. These results are consistent with our overall hypothesis that NzO impairs hypothalamic control of gonadotropin release, either by directly acting at the level of GnRH neurons or by altering, in particular, upstream events such as endogenous opioid and catecholaminergic neural influences on GnRH neurons.

Until recently, it was assumed that N20 acts non-specifically by altering membrane dynamics due to its lipid solubility. Recent studies indicate that N20, a small inorganic gas molecule, may act on specific receptors to induce anesthesia and other effects. For example, N-methyl-D-aspartate (NMDA) receptors are inhibited by N20. Exposure to N20 in intact animals also has been shown to selectively increase mRNA expression for the alpha-1 subunit of GABAA receptors in discrete regions of the central nervous system. A recent study indicates that N20 selectively elevates mRNA levels for GABAA receptors in the brains of mice and enhances GABA-induced chloride influx. Furthermore, N20 may also influence G-protein-cou-pled potassium channels.

Because of these varied actions of N20 on membrane receptors, and because extracellular signals are transduced to the nucleus via a variety of cell signaling mechanisms to alter gene expression, we also studied the effects of NzO on some cell signaling pathways in GT1-7 cells. We selected only a few cell signaling pathways. Because N20 has been reported to influence G-protein-coupled potassium channels, we examined the effect of the gas on intracellular pathway downstream of G-protein-coupled receptor signaling, the p44/42 pathway to transcription regulation. In this pathway, activated p44/42 dimer can regulate targets in cytoplasm and also translocate to the nucleus where it phosphor-ylates a number of transcription factors to regulate gene expression. We also explored the pathway responsible for inhibition of apoptosis because GT1-7 cells maintained under 60% N20 appeared healthy and numerous (Figure IB). Inhibitor of nuclear factor kappa В (IKB) inhibits apoptosis signaling via nuclear factor kappa В (NF-кВ). An increase in IKB activity would indicate a protective effect against apoptosis. The mitogen-acti-vated protein kinases (eg, p38) are involved in many cellular programs such as cell proliferation, cell differentiation, and cell death.
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Finally, c-fos is one of several transcription factors regulating gene expression, and has been shown to be associated with GnRH promoter activity. Cell signaling activation is a rapid process, hence we exposed the cells for shorter durations of time to N20 (typically 10180 minutes). The c-fos activity was examined at several hours of exposure to N20 because GnRH mRNA levels were changed only after 24 hours of exposure to NzO. We explored the effects of N20 phosphorylation of p44/42, p63, and p38 as well as c-fos and IKB. Our results indicate that N20 short-term exposure does not significantly change phosphorylation of ERK1/2, p38, or IKB. In addition, we did not detect any changes in c-fos levels after N20 exposure. However, N20 exposure resulted in a significant increase in p63 phosphorylation in the GT1-7 cells, indicating that N20 may influence certain phosphorylation events within the cell whose significance is not clear from this study. Whether these changes in a 63,000 Dalton protein (p63) phosphorylation are somehow linked to decreased GnRH mRNA levels requires further investigation. Also, the long-term effects of exposure to N20 on signaling mechanisms such as those regulated by МАРК, PKC, and cAMP are not known at present. We are developing a perfusion system using N20 dissolved in the medium to study the effects of N20 on signal transduction events in order to understand the cellular mechanisms of action of its effects on GnRH gene transcription.

In conclusion, we demonstrated for the first time that N20 exposure is associated with a drastic reduction in the levels of GnRH mRNA along with a decreased ability of GnRH secretion by the GT1-7 cells. silagra uk

N20 is one of the most widely used anesthetic agents in dental medicine. Occupational hazards associated with unscavenged N20 exposure are a significant problem in the human population because more than 250,000 female dental assistants, 148,000 dental hy-gienists, and 25,000 female dentists are directly associated with dental practice in the United States alone. In many states, N20 remains unregulated. Animal and human studies indicate that N20 may have adverse effects on reproductive function, but the mechanisms of action on the neuroendocrine components regulating reproductive function remain largely unexplored. Although it is difficult to disassociate the direct effects of N20 on GnRH neurons from its indirect effects because of complex multineuronal regulation of GnRH neurons in the live animal, in vivo studies are essential in order to draw definitive conclusions on the overall effects of N20 on GnRH cell function. In vitro methods employed in this study have limitations. Although results support the hypothesis of a direct effect of this gas anesthetic on the GnRH neurons, in vivo studies must be performed with low levels of N20 before extrapolating these results to animals or humans.

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