An assessment of the effects of selected antiretroviral drugs on steroid hormone production using an in vitro steroidogenesis assay
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Various contaminants already exist in the environment, with knowledge of more emerging each day. Some of these emerging contaminants are pseudo-persistent. Examples of these are pharmaceuticals and personal care products (PPCPs). PPCPs are designed to have a biological effect at very low doses, and therefore they are capable of causing a lot of harm in the environment. Due to the high prevalence of human immunodeficiency virus in South Africa, there are copious amounts of antiretroviral (ARV) treatments being administered. The ARVs that are unused or not fully metabolised make their way into the receiving environment, inter alia via wastewater treatment plants. ARVs along with many other PPCPs have already been detected in various water sources globally. However, the effects that these environmental ARVs may have on various non-target organisms, is not well researched. Various PPCPs are known to be endocrine disruptive in vitro. Therefore, this study aimed to determine the effect of known environmentally relevant concentrations of ARV active ingredients on steroidogenesis in vitro. The H295R human adrenocortical carcinoma cells were exposed to six concentrations (between 0.0008 and 80 ng/L) of six ARVs for 48 hours. The change in hormone concentrations in the nutrient growth medium was compared to that of solvent exposed control cells and expressed as a fold-change. Data was corrected for the evaporation of nutrient media during exposure, as well as the viability of the cells. The six hormones quantified were testosterone, 17β-oestradiol, aldosterone, cortisol, androstenedione, and 17α-hydroxyprogesterone. The method to quantify six steroid hormones was developed and validated for the purpose of this study using an ultra-high-pressure liquid chromatograph, quadrupole time-of-flight mass spectrometer. Results were variable and not always dose-dependent: a single ARV would decrease hormone levels at high concentrations, just to increase again at a lower concentration, followed by an increase again at an even lower concentration. In general, lopinavir, lamivudine, stavudine and efavirenz exposures decreased 17β-oestradiol levels, while ritonavir increased 17β-oestradiol levels. Testosterone levels decreased with exposures to ritonavir, lamivudine, stavudine and efavirenz. Furthermore, ritonavir, efavirenz and stavudine exposures resulted in decreased cortisol levels, while stavudine, didanosine and efavirenz decreased androstenedione. 17α-hydroxyprogesterone decreased with efavirenz and stavudine exposures, but increased with exposures to ritonavir, lopinavir, didanosine and lamivudine. Therefore, the results from this study show that all the ARVs tested influenced the steroidogenesis process in the H295R cells to some extent, possibly causing endocrinedisrupting effects in vertebrate organisms exposed to concentrations occurring in the natural environment.