Neuroactive steroids are synthesized de novo in brain, yet their physiological significance remains elusive. We provide biochemical, electrophysiological, and behavioral evidence that several specific actions of alcohol (ethanol) are mediated by the neurosteroid 3␣-hydroxy-5␣-pregnan-20-one (3␣,5␣-THP; allopregnanolone). Systemic alcohol administration elevates 3␣,5␣-THP levels in the cerebral cortex to pharmacologically relevant concentrations. The elevation of 3␣,5␣-THP is doseand time-dependent. Furthermore, there is a significant correlation between 3␣,5␣-THP levels in cerebral cortex and the hypnotic effect of ethanol. Blockade of de novo biosynthesis of 5␣-reduced steroids using the 5␣-reductase inhibitor finasteride prevents several effects of ethanol. Pretreatment with finasteride causes no changes in baseline bicuculline-induced seizure threshold but reverses the anticonvulsant effect of ethanol. Finasteride pretreatment also reverses ethanol inhibition of spontaneous neural activity in medial septal/diagonal band of Broca neurons while having no direct effect on spontaneous firing rates. Thus, elevation of 3␣,5␣-THP levels by acute ethanol administration represents a novel mechanism of ethanol action as well as an important modulatory role for neurosteroids in the CNS.

This study explores the hypotheses that: (1) ethanol will interact with dl-Methylphenidate (MPH) to enantioselectively elevate plasma d-MPH, and primarily yield l-ethylphenidate as a transesterification metabolite; (2) women will exhibit lower relative bioavailability of MPH than men; and (3) sex-dependent differences in subjective effects will exist. dl-MPH HCl (0.3 mg/kg) was administered orally 30 min before ethanol, 30 min after ethanol (0.6 gm/kg), or without ethanol, in a randomized, normal subject three-way crossover study of 10 men and 10 women. Pharmacokinetic parameters were compared. Subjective effects were recorded using visual analog scales. One subject was a novel poor MPH metabolizer whose data were analyzed separately. Ethanol after or before MPH significantly (P<0.0001) elevated the geometric mean for the maximum d-MPH plasma concentration (C max (±SD)) from 15.3 (3.37) ng/ml to 21.5 (6.81) and 21.4 (4.86), respectively, and raised the corresponding geometric mean for the area under the concentration-time curve values from 82.9 (21.7) ng ml/h to 105.2 (23.5) and 102.9 (19.2). l-MPH was present in plasma only at 1-3% of the concentration of d-MPH, except in the poor metabolizer where l-MPH exceeded that of d-MPH. The metabolite l-ethylphenidate frequently exceeded 1 ng/ml in plasma, whereas d-ethylphenidate was detected only in low pg/ml concentrations. Women reported a significantly greater stimulant effect than men when questioned "Do you feel any drug effect?" (P<0.05), in spite of lower mean plasma d-MPH area under the response-time curves in women. Ethanol elevates plasma d-MPH C max and area under the concentration-time curve by approximately 40% and 25%, respectively. If the poor metabolizer of MPH proves to be a distinct phenotype, determining the genetic mechanism may be of value for individualizing drug therapy. The more pronounced stimulant effects experienced by women have sex-based abuse liability implications. NIH Public Access Author ManuscriptClin Pharmacol Ther. Author manuscript; available in PMC 2011 October 6. RESULTS Human subjectsTwenty research subjects (10 men aged 23-40 years: mean (±SD) 28.8 (5.3) years, weight 82.2 (10.5) kg, eight white, one black, one Hispanic; and 10 women aged 23-35 years: mean 28.7 (4.4) years, weight 65.2 (8.4) kg, nine white, one black, completed the entire protocol. One additional subject declined further participation after her first visit, citing discomfort with blood sampling. This subject was replaced with another female volunteer to ensure 10 of each sex completed the study. No adverse events occurred that were attributable to MPH, ethanol, or a combination thereof. All vital signs remained within normal parameters. Finally, no subject had any clinically significant findings on poststudy "exit" laboratory tests. (Figure 3). MPH-ethanol pharmacokinetic interactions Sex differences in d-MPH-ethanol pharmacokineticsThe mean (SD) extent of exposure (AUC) of d-MPH was significantly (P=0.042) greater in men (93.4 (25.3) ng h/ml) than i...

Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations and potentially contributing to the development of cancer. Because of their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples, including immunoassay, HPLC, and 32P-postlabeling, isotope dilution high performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adduct concentrations in biological samples are between 0.01–10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry, especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS, have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke.

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