Microplastics analytics: why we should not underestimate the importance of blank controls.

Recent years have seen considerable scientific attention devoted towards documenting the presence of microplastics (MPs) in environmental samples. Due to omnipresence of environmental microplastics, however, disentangling environmental MPs from sample contamination is a challenge. Hence, the environmental (collection site and laboratory) microplastics contamination of samples during processing is a reality that we must address, in order to generate reproducible and reliable data. Here we investigated published literature and have found that around 1/5 of studies failed to use blank controls in their experiments. Additionally, only 34% of the studies used a controlled air environment for their sample processing (laminar flow, fume hood, closed laboratory, clean room, etc.). In that regard, we have also shown that preparing samples in the fume hood, leads to more microplastics > 1 µm) contamination than preparing it in the laboratory bench and the laminar flow. Although it did not completely prevent microplastics contamination, the processing of sample inside the laminar flow is the best option to reduce sample contamination during processing. Overall, we showed that blank controls are a must in microplastics sample preparation, but it is often overlooked by researchers. Supplementary Information: The online version contains supplementary material available at 10.1186/s43591-023-00065-3.

The Analgesic Dipyrone Affects Pregnancy Outcomes and Endocrine-Sensitive Endpoints in Female and Male Offspring Rats.

Dipyrone is an analgesic and antipyretic drug commonly used in many countries. Although generally not recommended during pregnancy, it is known that many women use dipyrone during the gestational period. In this study, we investigated the endocrine and reproductive effects of dipyrone in female and male offspring rats exposed in utero from gestational days 10-21. Pregnant rats were treated with dipyrone at 25, 75, and 225 mg/kg/day via oral gavage. Developmental landmarks-anogenital index (AGI), number of nipples, vaginal opening, first estrus, and preputial separation-were evaluated in the offspring. Reproductive parameters, including estrous cycle regularity, daily sperm production, weight and histopathology of reproductive organs, steroid hormone levels, and gene expression of selected markers of reproductive function were assessed at adulthood. At the highest dose, dipyrone induced a significant increase in postimplantation losses/fetal death and delayed parturition in dams. Offspring exposed in utero to the highest dose also exhibited significant changes in some early life markers of endocrine disruption, in particular increased AGI in females, indicating a proandrogenic effect, and increased rate of retained nipples in males, indicating an antiandrogenic response. No changes were observed in markers of puberty onset or reproductive parameters at adulthood. These results suggest that exposure to therapeutically relevant doses of dipyrone may induce mild endocrine disruptive effects that can be detected in late pregnancy and early life. Such effects may be relevant considering dipyrone use by pregnant women and the possibility of coexposures with other endocrine disruptors.

Uterotrophic and in vitro screening for (anti)estrogenic activity of dipyrone.

Dipyrone is a commonly used analgesic in many countries and there is limited data on its possible endocrine disrupting effects. We performed a screening for in vivo and in vitro anti(estrogenic) activity of dipyrone. For the in vivo uterotrophic assay, immature female rats (22-days-old) were treated daily by oral gavage for three days with different doses of dipyrone alone (50, 100, 200 mg/kg/day) and associated with three ethynylestradiol (EE) doses (1, 3 and 10 µg/kg/day), which were based on a dose-response curve experiment. The uterine weight was used as a biomarker for estrogenicity. In a parallel in vitro approach, we used a yeast-based transcriptional activation reporter gene assay (Yeast Estrogen Screening - YES) for assessment of estrogenic agonistic and antagonistic effects of dipyrone and its main metabolites 4-methylaminoantipyrine (MAA) and 4-aminoantipyrine (AA). In the uterotrophic assay, animals that received EE at 1, 3 and 10 µg/kg/day showed an increase in relative uterine weight compared with vehicle-only rats (canola oil). Dipyrone did not increase uterine weight at any dose tested (50, 100 and 200 mg/kg/day) in relation to vehicle control, indicating absence of estrogenic activity. Furthermore, co-administration of dipyrone (50 and 200 mg/kg/day) and EE (1, 3 or 10 µg/kg/day) was unable to block EE estrogenic action in comparison to the groups treated with EE alone, indicating absence of antiestrogenic activity. In the YES assay dipyrone and its metabolites did not demonstrate estrogen agonistic or antagonistic properties in the yeast cells. These results suggest that dipyrone and its metabolites do not produce (anti)estrogenic effects in vivo or in vitro.

Prenatal diclofenac exposure delays pubertal development and induces behavioral changes in rats.

Diclofenac is a non-steroidal anti-inflammatory drug widely used by the general population and, although generally contraindicated during pregnancy, it is also used by some pregnant women. This study investigated endocrine, reproductive and behavioral effects of diclofenac in male and female offspring rats exposed in utero from gestational days 10-20. Pregnant rats were treated with diclofenac at doses of 0.2, 1 and 5 mg/kg/day via oral gavage. Anogenital distance (AGD), number of nipples, and developmental landmarks of puberty onset - vaginal opening (VO), first estrus (FE) and preputial separation (PPS) - were evaluated in the offspring. At adulthood, behavioral and reproductive parameters were assessed. Male and female rats were tested in the elevated plus maze test to assess locomotor activity and anxiety-like behaviors, while male rats were also evaluated in the partner preference test. No significant effects were observed on AGD and number of nipples in both males and females. Diclofenac treatment induced an overall delay in developmental landmarks of puberty onset in male and female offspring, which reached statistical significance for PPS at the lowest diclofenac dose. Prenatal exposure to all tested doses abolished the preference of male rats for an estrous female, suggesting an impairment of brain masculinization. No changes were observed on male or female reproductive parameters at adulthood. Overall, our results indicate that prenatal exposure to therapeutically relevant doses of diclofenac may have an impact in the pubertal development of rats and negatively affect male partner preference behavior.

In Utero and Lactational Exposure to Diisopentyl Phthalate Induces Fetal Toxicity and Antiandrogenic Effects in Rats.

A previous study has demonstrated exposure of Brazilian pregnant women to diisopentyl phthalate (DiPeP), which reduces fetal rat testosterone production in a dose-responsive manner. In this study, we examined gene expression of steroidogenic proteins in rat fetal testes and investigated the effects of in utero and lactational DiPeP exposure on male rat reproductive development and function. For the prenatal experiment, we orally exposed pregnant Wistar rats to DiPeP or di-n-butyl phthalate (reference phthalate) at 0, 125, 250, and 500 mg/kg/day from gestation day 14-18 and the fetal testis was evaluated for transcript expression of Star, Cyp11a1, Cyp17a1, Cyp19a1, Insl3, Ar, Esr1, Esr2, and Gper1 by real-time quantitative PCR (RT-qPCR). Diisopentyl phthalate lowered mRNA levels of key steroidogenic proteins, lending support to the previously reported reductions in fetal testosterone production. Diisopentyl phthalate also lowered fetal testis transcript levels of Insl3 and changed gene expression of some steroid hormones receptors. For the postnatal experiment, pregnant rats were exposed orally to vehicle (canola oil) and 4 DiPeP doses (1, 10, 100, and 300 mg/kg/day) between gestation day 10 and postnatal day 21. Diisopentyl phthalate induced a range of reproductive and antiandrogenic effects that are typical of the rat phthalate syndrome, including reduced anogenital distance at the highest dose, reduced weight of seminal vesicles at 10 mg/kg/day and above, and testicular morphological and functional changes. Signs of fetal toxicity were observed at the highest dose. Together, our results indicate that DiPeP, a compound relevant to the human exposure scenario, is one of the most active antiandrogenic phthalates.

Assessment of the analgesic dipyrone as a possible (anti)androgenic endocrine disruptor.

Mild analgesics have been associated with antiandrogenic effects, but there are no such studies on dipyrone, despite its high prevalence of use in many countries. We examined the production of steroid hormones in human H295R cells after exposure to dipyrone and two metabolites, 4-Methylaminoantipyrine (MAA) and 4-Aminoantipyrine (AA), as well as fetal testicular testosterone production in rats following maternal dipyrone exposure. Androgen agonistic/antagonistic effects were examined in vitro for dipyrone and its metabolites in the Yeast Androgen Screen (YAS) assay and in vivo for dipyrone through the Hershberger assay. In vitro we tested dipyrone, MAA, and AA (0.1-1000 µM) while in vivo we used dipyrone (50, 100, 200 mg/kg/day). In the H295R assay, dipyrone, MAA and AA reduced the production of androgens and corticosteroids. Testosterone was reduced at concentrations 4-13 times higher than the maximum plasma concentrations reported in humans for MAA and AA. No effects were observed in the fetal testosterone production assay. In the YAS and Hershberger assays, no androgen agonistic/antagonistic activities were observed. These results indicate that dipyrone and its metabolites do not interact with the androgen receptor, but have the potential to inhibit steroidogenesis, however only at concentrations that are not relevant under normal medical use.