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.

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Viability
Exposure to 60% N20 over a period of 24 hours did not affect viability of the GT1-7 cells (Figure 1A). The total protein content of the cells was also not significantly different between air-exposed and N20-exposed cells (Figure 1B). The effects of oxygen concentration (21% vs 35%) on cell viability and protein concentration over 36 hours were also evaluated in a series of preliminary studies. Thirty-five percent oxygen concentration did not have any significant effects on these measures.

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Culture of GT1-7 Cell Line
GT1-7 cells (gift of Dr P.M. Mellon, University of California, San Diego) were maintained in DMEM-4.5 g/L glucose (Life Technologies, Inc, Rockville, MD) supplemented with 10% fetal calf serum, 100 units/mL generic penicillin, 100 mg/mL streptomycin, and 0.0025 mg/mL amphotericin В at 37°C in a humidified atmosphere of 5% C02, 95% air or 60% N20, 35% 02, and 5% C02. For performing various tests, cells were plated in 100mm tissue culture dishes. Experiments were typically performed at day 3 of plating cells onto the tissue culture dishes.

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Since its first use in 1844, nitrous oxide (N20) has been considered one of the safest anesthetics and is still widely used in clinical practice. However, a number of studies have associated chronic exposure to N20 with certain adverse effects, such as leukopenia, embryo toxicity, and fetal death. Exposure to N20 in operating rooms appears to result from passive inhalation of the gas. Epidemiologic investigations conducted in the early 1970s suggested that significant occupational hazards to female personnel (birth defects and abortions) were associated with long-term employment in operating rooms using NjpO. In addition, exposure to N20 in facilities where the gas is not scavenged may also result in a sig nificant decrease in fertility in women. Supporting evidence from animal studies indicates that subclinical concentrations of N20 over time result in fertility problems in both male and female rats as well as decreased litter size and weight of the Fx generation.

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