Identification of sex pheromone of red swamp crayfish Procambarus clarkii and exploration of the chemosensory mechanism of their antennae.

Red swamp crayfish, Procambarus clarkii, is a globally invasive species, which has caused great damage to biodiversity, agriculture, and fishing. Therefore, the development of effective management methods, such as pheromone control, is necessary for biological control and biodiversity protection. However, the components of P. clarkii sex pheromones have not yet been explored, and the chemosensory mechanism of the P. clarkii antennae after stimulation by sex pheromone also remains unknown. In this study, we isolated and identified the candidate bioactive component of the female P. clarkii sex pheromone using ultrafiltration centrifugation, semi-preparative liquid phase separation and omics technologies and conducted bioassays to determine its attraction ability. Meanwhile, RNA-Seq technology was used to analyze the potential chemosensory mechanism of antennae. Our results indicated that the male P. clarkii were uniaxially attracted to the female crude conditioned water (FCW), medium fraction (MF, isolated by ultrafiltration centrifugation), and preparative fragment 6 of females (PFF6, isolated by semi-preparative liquid phase separation). Metabolomic analysis revealed the presence of 18 differential metabolites between the PFF6 and PFM6 samples, among which 15 were significantly upregulated in the PFF6 sample. Bioassay test also showed that mestranol, especially at concentrations of 10-5-10-2 mol∙l-1, could significantly attract P. clarkii males; therefore, mestranol was identified as the candidate sex pheromone component of P. clarkii females. Furthermore, RNA-Seq results showed that most differentially expressed genes (DEGs) enriched in lipid metabolism and signal transduction pathways were up-regulated in P. clarkii males. In addition, high expressions of Ca2+-binding protein and ion transporting ATPases may enhance the sensitivity of the antennae of P. clarkii males towards sex pheromones. Our study provides data on P. clarkii sex pheromone composition and reveals the molecular mechanism of sex pheromone response in P. clarkii. Moreover, our study provides a referable method for the isolation of candidate bioactive molecules from the P. clarkii sex pheromone.

Effects of low-dose bisphenol AF on mammal testis development via complex mechanisms: alterations are detectable in both infancy and adulthood.

Despite growing concern about adverse effects of bisphenol AF (BPAF) due to its endocrine disrupting properties, there is a lack of toxicity data from low-dose studies and direct evidence linking its adverse effects to endocrine disrupting properties. Here, we investigated the effects of gestational and postnatal exposure to BPAF through drinking water (0.15-15 µg/mL, equivalent to the daily intake of ~ 50 and 5 mg/kg/day) on testis development in mice. We found that like mestranol, 5 mg/kg/day BPAF resulted in remarkable decreases in multiple male reproductive parameters in adulthood, such as the sperm number and serum testosterone level. Notably, 50 µg/kg/day BPAF also caused significant decreases in anogenital distance (AGD), the luteinizing hormone level and spermatocyte number, along with declining trends in sperm number and the serum levels of testosterone and follicle-stimulating hormone. In line with the adverse outcomes observed in adulthood, on postnatal day (PND) 9, we also observed BPAF-caused dose-dependent alterations, including reduced AGD, seminiferous tubule area and numbers of total germ cells, spermatocytes and Leydig cells, coupled with down-regulated expression of male-biased genes in testes. Even when exposure to 5 mg/kg/day BPAF as well as MES was initiated from PND 0, similar alterations in male reproductive parameters were also found on PND 9, along with a decrease in the GnRH content in the hypothalamus; moreover, testicular alterations and the reduction in AGD were partly antagonized by the estrogen receptor (ER) antagonist ICI 182,780, but the reduction of GnRH production was not done, showing that the effects of BPAF on testis development may be partially mediated by ER signaling. In conclusion, all the findings demonstrate that low-dose BPAF can partly disrupt mammal testis development and cause adverse testicular outcomes in adulthood, indicating a potential reproductive risk to mammals including humans. Importantly, our finding that developmental alterations elicited by BPAF have been detectable on PND 9 provides important motivation for the development of effective methods for early detection of adverse effects of estrogenic chemicals on testis development.

Effect-directed analysis of a hospital effluent sample using A-YES for the identification of endocrine disrupting compounds.

An effect-directed analysis (EDA) approach was used to identify the compounds responsible for endocrine disruption in a hospital effluent (Basque Country). In order to facilitate the identification of the potentially toxic substances, a sample was collected using an automated onsite large volume solid phase extraction (LV-SPE) system. Then, it was fractionated with a two-step orthogonal chromatographic separation and tested for estrogenic effects with a recombinant yeast (A-YES) in-vitro bioassay. The fractionation method was optimized and validated for 184 compounds, and its application to the hospital effluent sample allowed reducing the number of unknowns from 292 in the raw sample to 35 after suspect analysis of the bioactive fractions. Among those, 7 of them were confirmed with chemical standards. In addition, target analysis of the raw sample confirmed the presence of mestranol, estrone and dodemorph in the fractions showing estrogenic activity. Predictive estrogenic activity modelling using quantitative structure-activity relationships indicated that the hormones mestranol (5840 ng/L) and estrone (128 ng/L), the plasticiser bisphenol A (9219 ng/L) and the preservative butylparaben (1224 ng/L) were the main contributors of the potential toxicity. Derived bioanalytical equivalents (BEQs) pointed mestranol and estrone as the main contributors (56 % and 43 %, respectively) of the 50 % of the sample's explained total estrogenic activity.

Oral contraceptive use by formulation and endometrial cancer risk among women born in 1947-1964: The Nurses' Health Study II, a prospective cohort study.

Oral contraceptives (OCs) have been associated with long-term lower endometrial cancer risk; relatively little is known about associations with more recent OC formulations and associations with longer-term risk. A total of 107,069 women from the Nurses' Health Study II recalled OC use from age 13 to baseline (1989); biennial questionnaires updated data on OC use until 2009. OCs were classified by estrogen and progestin type, dose, and potency based on reported brand. 864 incident endometrial cancer cases were identified through 2017. Multivariable Cox proportional hazards models estimated hazard ratios (HR) and 95% confidence intervals [95% CI] for the association of OC use with endometrial cancer risk. OC use was associated with lower endometrial cancer risk (ever use, HR 0.77 [95% CI 0.65-0.91]; >10 years of use, 0.43 [0.32-0.58] vs. never OC use). Inverse associations for duration were evident regardless of time since last use. Longer durations (> 5 years) of ethinyl estradiol (0.52 [0.41-0.67]) and second-generation progestins (0.43 [0.30-0.61]), both versus never use, were more strongly associated with lower risk than mestranol (0.66 [0.50-0.88], p-het = 0.01) and first-generation progestins (0.62 [0.49-0.78], p-het = 0.03). Inverse associations were generally observed for cross-classified cumulative average estrogen and progestin dose and potency (< vs. ≥ median; ever use vs. never OC use), with the exception of high estrogen and low progestin dose. OCs were associated with lower endometrial cancer risk, independent of time since last use. Use of ethinyl estradiol and second-generation progestins were more strongly inversely associated with risk compared with older formulations.

Evaluation of antimicrobial efficacy of Trachyspermum ammi (Ajwain) oil and chlorhexidine against oral bacteria: An in vitro study.

INTRODUCTION: Plaque removal is of utmost importance for control of dental caries and other associated diseases of oral cavity. However, various natural agents have proven their efficacy over chemotherapeutic agents in terms of antibacterial activity against various microorganisms. The effect is mainly due to polyphenol as its major constituent. AIM: In this in vitro study, we aimed to determine the antibacterial efficacy of Trachyspermum ammi oil at different concentrations against five oral bacteria. HYPOTHESIS: Herbal compound, T. ammi oil is effective in reducing five oral plaque-forming bacteria. MATERIALS AND METHODS: We determined the antimicrobial activity of T. ammi oil (test material) against chlorhexidine (gold standard). Pure cultures of Streptococcus mutans MTCC No 497, Streptococcus oralis MTCC No. 2696, Lactobacillus acidophilus MTCC No. 10307, Lactobacillus fermentum MTCC No. 903, and Candida albicans MTCC No. 183 were obtained and grown in selective culture media. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of both materials were evaluated by serial dilution and disc diffusion method, respectively. RESULTS: Our results revealed that T. ammi oil moderately inhibits bacterial growth with mean MIC of 250, 125, 250, 125, and 250 µg/ml, respectively. Mean MBC for T. ammi oil obtained was 18.60 ± 0.65, 11.60 ± 1.14, 14.10 ± 0.55, 11.50 ± 0.61, and 15.10 ± 0.74 mm. The MIC and MBC values were higher as compared to chlorhexidine gluconate and it was statistically significant. CONCLUSION: T. ammi (ajwain) can serve as a potential, natural, nontoxic, and economical therapeutic antiplaque agent.

Parallel Solid-Phase Synthesis using a New Diethylsilylacetylenic Linker and Leading to Mestranol Derivatives with Potent Antiproliferative Activities on Multiple Cancer Cell Lines.

BACKGROUND: RM-133 belongs to a new family of aminosteroid derivatives demonstrating interesting anticancer properties, as confirmed in vivo in four mouse cancer xenograft models. However, the metabolic stability of RM-133 needs to be improved. After investigation, the replacement of its androstane scaffold by a more stable estrane scaffold led to the development of the mestranol derivative RM-581. METHODS: Using solid-phase strategy involving five steps, we quickly synthesized a series of RM-581 analogs using the recently-developed diethylsilylacetylenic linker. To establish structure-activity relationships, we then investigated their antiproliferative potency on a panel of cancer cell lines from various cancers (breast, prostate, ovarian and pancreatic). RESULTS: Some of the mestranol derivatives have shown in vitro anticancer activities that are close to, or better than, those observed for RM-581. Compound 23, a mestranol derivative having a ((3,5-dimethylbenzoyl)- L-prolyl)piperazine side chain at position C2, was found to be active as an antiproliferative agent (IC50 = 0.38 ± 0.34 to 3.17 ± 0.10 µM) and to be twice as active as RM-581 on LNCaP, PC-3, MCF-7, PANC-1 and OVCAR-3 cancer cells (IC50 = 0.56 ± 0.30, 0.89 ± 0.63, 1.36 ± 0.31, 2.47 ± 0.91 and 3.17 ± 0.10 µM, respectively). CONCLUSION: Easily synthesized in good yields by both solid-phase organic synthesis and classic solution-phase chemistry, promising compound 23 could be used as an antiproliferative agent on a variety of cancers, notably pancreatic and ovarian cancers, both having very bad prognoses.

A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance.

This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R2 of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants.

Design of a Mestranol 2-N-Piperazino-Substituted Derivative Showing Potent and Selective in†vitro and in†vivo Activities in MCF-7 Breast Cancer Models.

Anticancer structure-activity relationship studies on aminosteroid (5α-androstane) derivatives have emerged with a promising lead candidate: RM-133 (2ß-[1-(quinoline-2-carbonyl)pyrrolidine-2-carbonyl]-N-piperazine-5α-androstane-3α,17ß-diol), which possesses high in vitro and in vivo activities against several cancer cells, and selectivity over normal cells. However, the relatively weak metabolic stability of RM-133 has been a drawback to its progression toward clinical trials. We investigated the replacement of the androstane backbone by a more stable mestranol moiety. The resulting compound, called RM-581 ({4-[17α-ethynyl-17ß-hydroxy-3-methoxyestra-1,3,5(10)-trien-2-yl]piperazin-1-yl}[(2S)-1-(quinolin-2-ylcarbonyl)pyrrolidin-2-yl]methanone), was synthesized efficiently in only five steps from commercially available estrone. In comparison with RM-133, RM-581 was found to be twice as metabolically stable, retains potent cytotoxic activity in breast cancer MCF-7 cell culture, and fully blocks tumor growth in a mouse xenograft model of breast cancer. Advantageously, the selectivity over normal cells has been increased with this estrane version of RM-133. In fact, RM-581 showed a better selectivity index (15.3 vs. 3.0) for breast cancer MCF-7 cells over normal breast MCF-10A cells, and was found to be nontoxic toward primary human kidney proximal tubule cells at doses reaching 50 µm.

Sterol ratios as a tool for sewage pollution assessment of river sediments in Serbia.

In this work, source pollution tracing of the sediments of the Danube River and its tributaries in Serbia was performed using sterol ratios. Improved liquid chromatography-tandem mass spectrometry method, which enabled complete chromatographic separation of four analytes with identical fragmentation reactions (epicoprostanol, coprostanol, epicholestanol and cholestanol), was applied for the determination of steroid compounds (hormones, human/animal and plant sterols). A widespread occurrence of sterols was identified in all analyzed samples, whereas the only detected hormones were mestranol and 17α-estradiol. A human-sourced sewage marker coprostanol was detected at the highest concentration (up to 1939 ng g(-1)). The ratios between the key sterol biomarkers, as well as the percentage of coprostanol relative to the total sterol amount, were applied with the aim of selecting the most reliable for distinction between human-sourced pollution and the sterols originated from the natural sources in river sediments. The coprostanol/(cholesterol + cholestanol) and coprostanol/epicoprostanol ratios do not distinguish between human and natural sources of sterols in the river sediments in Serbia. The most reliable sterol ratios for the sewage pollution assessment of river sediments in the studied area were found to be coprostanol/(coprostanol + cholestanol), coprostanol/cholesterol and epicoprostanol/coprostanol. For the majority of sediments, human-derived pollution was determined. Two sediment samples were identified as influenced by a combination of human and natural biogenic sources.

Effect of CYP2C9 Genetic Polymorphism in a Chinese Population on the Metabolism of Mestranol in vitro.

BACKGROUND: Mestranol is a widely used estrogen, which is converted into its active metabolite ethinyl estradiol by cytochrome P450 (CYP) 2C9. To comprehensively examine the enzymatic activity of reported CYP2C9 variants in Chinese individuals in response to mestranol, wild-type CYP2C9*1 and 35 allelic variants were highly expressed in Sf21 insect cell microsomes and used for the detection of their enzymatic values in vitro. These results showed that the majority of tested variants exhibited decreased clearance values compared to wild type, except for CYP2C9*40 and *36. METHOD: Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.25-8 µmol/l mestranol for 30 min at 37°C. Then, the production of the metabolite of mestranol, ethinyl estradiol, was analyzed using high-performance liquid chromatography. RESULTS: Most CYP-catalyzed reactions were sufficiently described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), while 9 variants exhibited atypical or non-Michaelis-Menten kinetic values, which were largely due to the self-inhibitory effect in response to mestranol. CONCLUSION: This is the first report of these rare alleles for mestranol metabolism, which provides fundamental data for further clinical studies on CYP2C9 alleles for mestranol metabolism.

Amphidynamic crystals of a steroidal bicyclo[2.2.2]octane rotor: a high symmetry group that rotates faster than smaller methyl and methoxy groups.

The synthesis, crystallization, single crystal X-ray structure, and solid state dynamics of molecular rotor 3 provided with a high symmetry order and relatively cylindrical bicyclo[2.2.2]octane (BCO) rotator linked to mestranol fragments were investigated in this work. By use of solid state (13)C NMR, three rotating fragments were identified within the molecule: the BCO, the C19 methoxy and the C18 methyl groups. To determine the dynamics of the BCO group in crystals of 3 by variable temperature (1)H spin-lattice relaxation (VT (1)H T1), we determined the (1)H T1 contributions from the methoxy group C19 by carrying out measurements with the methoxy-deuterated isotopologue rotor 3-d6. The contributions from the quaternary methyl group C18 were estimated by considering the differences between the VT (1)H T1 of mestranol 8 and methoxy-deuterated mestranol 8-d3. From these studies it was determined that the BCO rotator in 3 has an activation energy of only 1.15 kcal mol(-1), with a barrier for site exchange that is smaller than those of methyl (E(a) = 1.35 kcal mol(-1)) and methoxy groups (E(a) = 1.92 kcal mol(-1)), despite their smaller moments of inertia and surface areas.

Synthesis, characterization and biological evaluation of some novel 17-isoxazoles in the estrone series.

Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isoxazoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moiety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding Δ(16,17)exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmacological studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7 and A2780).

Ultrasonic-assisted derivatization of estrogenic compounds in a cup horn booster and determination by GC-MS.

Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic-assisted derivatization step by means of a cup horn booster and the determination of estrone, 17beta-estradiol, estriol, 17alpha-ethynyl estradiol and mestranol was developed by GC-MS. Different derivatization reagents and solvents were studied for maximizing the di-derivatization of 17alpha-ethynyl estradiol under ultrasound energy. Only N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1-10 min), sonication power (20-80%), sonication cycles (1-9), derivatization reagent volume (25-125 microL) and solvent volume (25-125 microL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138-70%) and synthetic (112-89%)), the LODs (0.35-1.66 ng/L), and LOQs (1.16-5.52 ng/L) and the precision (0.2-5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.

The effect of etoricoxib on the pharmacokinetics of oral contraceptives in healthy participants.

The pharmacokinetics of oral contraceptive (OC) components, ethinyl estradiol (EE) and norethindrone (NET), were evaluated after coadministration with etoricoxib in 3 double-blind, randomized, 2-period crossover studies of healthy women. There were 16, 39, and 24 participants enrolled in studies 1 (part I, part II), and 2, respectively. Each participant received triphasic OC (EE 35 microg/NET 0.5 mgx7 days, 0.75 mgx7 days, 1.0 mgx7 days) throughout each 28-day period. OC was coadministered with 21 days of etoricoxib daily followed by placebo for 7 days; the alternate period followed the reverse regimen (placebo to etoricoxib). Study 1 (part I) examined concurrent (morning) administration of OC/etoricoxib 120 mg, study 1 (part II) examined staggered (morning/night) administration of OC/etoricoxib 120 mg, and study 2 examined concurrent (morning) administration of OC/etoricoxib 60 mg. Coadministration of OC and etoricoxib 120 mg once daily was associated with a approximately 50% to 60% increase in EE concentrations, whereas etoricoxib 60 mg once daily was associated with a approximately 37% increase in EE concentrations. Coadministration of OC and etoricoxib was generally well tolerated. A clinically important change in NET AUC0-24 h was not observed. Adverse events included dyspepsia, diarrhea, headache, nausea, fatigue, loss of appetite, and taste disturbance.

Improved synthesis of mestranol and ethinyl estradiol (EE) related degradation products as authentic references.

Preparative chemical methods for the synthesis of 10 degradation or photodecomposition products of mestranol and ethinyl estradiol (EE) are described. The synthesized compounds are useful as reference materials and standards for pharmaceutical analysis of mestranol and EE as bulk chemical or in formulated product. New synthetic methods were presented and the known synthetic procedures were improved. Detailed structural characterization of the degradation or photodecomposition products of mestranol and EE and related compounds was reported.

Microwave-accelerated derivatization for the simultaneous gas chromatographic-mass spectrometric analysis of natural and synthetic estrogenic steroids.

A rapid microwave-accelerated derivatization process for the GC-MS analysis of steroid estrogens, estrone (E1), 17beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), was developed. Under microwave irradiation, the five estrogenic hormones studied were simultaneously derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+trimethylchlorosilane (TMCS) in pyridine solution. Effects of irradiation time (15-120 s) and power level (240-800 W) on the yield of the derivatization were investigated. The derivatization under the irradiation of 800 W microwave for 60s produced comparable results when compared with the conventional heating process in a sand bath for 30 min at 80 degrees C in terms of derivatization yield, linearity and precision for all steroid hormones tested. The calibration curves are linear between 3.00 and 3.00 x 10(2) microg mL(-1). The square of the regression coefficients (R(2)) range from 0.979 to 1.000. The applicability of the method was evaluated on spiked river and distilled water samples at two concentrations, 25.0 and 2.00 x 10(2) ng mL(-1). The recoveries obtained by using microwave heating (60s, 800 W) were similar to those by conventional heating. When combined solid-phase extraction (SPE) with the application of the microwave-accelerated derivatization proposed here, the detection limits of 0.02-0.1 ng L(-1) for the steroid hormones have been achieved. The results demonstrated that microwave-accelerated derivatization is an efficient and suitable sample preparation method for the GC-MS analysis of estrogenic steroids.

Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies.

Endocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17beta-estradiol (E2) and different EDCs have been recently demonstrated to affect the function of the immune cells, the hemocytes. The effects were Tamoxifen-sensitive and were mediated by rapid modulation of kinase-mediated transduction pathways. In this work we compared the in vitro effects of individual estrogenic chemicals (E2, EE: 17alpha-ethynyl estradiol; MES: mestranol; NP: nonylphenol; NP1EC: nonylphenol monoethoxylate carboxylate; BPA: bisphenol A; BP: benzophenone) on hemocyte parameters: lysosomal membrane stability (LMS), phagocytosis, lysozyme release. LMS was the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. EC50 values obtained from LMS data were utilized to calculate the estradiol equivalency factor (EEF) for each compound; these EEFs allowed for an estimation of the estrogenic potential of a synthetic mixture with a composition very similar to that previously found in waters of the Venice lagoon. Concentrated mixtures significantly affected hemocyte parameters in vitro and the effects were prevented by Tamoxifen. Significant effects of the mixture were also observed in vivo, at longer exposure times and at concentrations comparable with environmental exposure levels. The results indicate that Mytilus immune parameters can be suitably utilized to evaluate the estrogenic potential of environmental samples.

The impact of sex and contraceptive therapy on the plasma and intracellular pharmacokinetics of zidovudine.

OBJECTIVES: Zidovudine remains part of combination antiretroviral therapy. Pharmacological studies rely on quantitation of active triphosphates in peripheral blood mononuclear cells. This study evaluated the impact of female sex and contraceptive therapy on zidovudine plasma and intracellular pharmacokinetics and the impact of contraceptive therapy on HIV viral load. METHODS: Serial plasma and intracellular zidovudine pharmacokinetics following oral and intravenous dosing were determined in 18 men and 20 women treated with zidovudine. Women could repeat pharmacokinetics assessment following 2 months oral or injectable contraceptive therapy. Zidovudine plasma and intracellular mono-, di- and triphosphate concentrations were determined by liquid chromatography tandem mass spectrometry. Plasma and cervical viral loads were determined preceding and following 2 months of contraceptive therapy in women. RESULTS: Men exhibited higher area under the concentration versus time curve for intracellular zidovudine and zidovudine-monophosphate following oral and intravenous dosing and higher zidovudine triphosphate following oral dosing. There was no difference between men and women in plasma zidovudine parameters. Furthermore, contraceptive therapy had no effect on zidovudine plasma or intracellular pharmacokinetics or on plasma or cervical HIV-1 RNA levels. CONCLUSIONS: Using an optimized pharmacokinetic design, this study indicated men exhibit significantly higher zidovudine-monophosphate and zidovudine-triphosphate exposure following zidovudine oral administration, having implications for drug toxicity and overall tolerance of zidovudine therapy. The lack of an effect of contraceptive therapy on zidovudine pharmacokinetics is surprising in light of previous pharmacokinetic studies for drugs eliminated primarily through glucuronidation. Contraceptive therapy had no effect on plasma or cervical viral load, results consistent with previous findings.