Toxicity Evaluation and Effect-Based Identification of Chlorine Disinfection Products of the Anti-COVID-19 Drug Chloroquine Phosphate.

Antiviral transformation products (TPs) generated during wastewater treatment are an environmental concern, as their discharge, in considerable amounts, into natural waters during a pandemic can pose possible risks to the aquatic environment. Identification of the hazardous TPs generated from antivirals during wastewater treatment is important. Herein, chloroquine phosphate (CQP), which was widely used during the coronavirus disease-19 (COVID-19) pandemic, was selected for research. We investigated the TPs generated from CQP during water chlorination. Zebrafish (Danio rerio) embryos were used to assess the developmental toxicity of CQP after water chlorination, and hazardous TPs were estimated using effect-directed analysis (EDA). Principal component analysis revealed that the developmental toxicity induced by chlorinated samples could be relevant to the formation of some halogenated TPs. Fractionation of the hazardous chlorinated sample, along with the bioassay and chemical analysis, identified halogenated TP387 as the main hazardous TP contributing to the developmental toxicity induced by chlorinated samples. TP387 could also be formed in real wastewater during chlorination in environmentally relevant conditions. This study provides a scientific basis for the further assessment of environmental risks of CQP after water chlorination and describes a method for identifying unknown hazardous TPs generated from pharmaceuticals during wastewater treatment.

Embryonic exposure to chloroxylenol induces developmental defects and cardiovascular toxicity via oxidative stress, inflammation, and apoptosis in zebrafish.

Chloroxylenol is an extensively consumed anti-microbial compound. Since its usage is on the rise due to the coronavirus pandemic and ban on other antimicrobial ingredients, recent studies have suggested the necessity of estimating its potential for ecotoxicity. The detrimental effect of chloroxylenol on zebrafish (Danio rerio) viability has been reported; however, research on the mechanisms underlying its toxicity is quite limited. Therefore, we applied the zebrafish model for elucidating responses against chloroxylenol to predict its toxicity toward human health and ecology. Zebrafish exposed to chloroxylenol (0, 0.5, 1, 2.5, 5, and 10 mg/L) at the embryonic stage (from 6 h post-fertilization (hpf) to 96 hpf) showed impaired viability and hatchability, and pathological phenotypes. To address these abnormalities, cellular responses such as oxidative stress, inflammation, and apoptosis were confirmed via in vivo imaging of a fluorescent dye or measurement of the transcriptional changes related to each response. In particular, developmental defects in the cardiovascular system of zebrafish exposed to 0, 0.5, 1, and 2.5 mg/L of chloroxylenol from 6 to 96 hpf were identified by structural analyses of the system in the flk1:eGFP transgenic line. Additional experiments were conducted using human umbilical vein endothelial cells (HUVECs) to predict the adverse impacts of chloroxylenol on the human vascular system. Chloroxylenol impairs the viability and tube formation ability of HUVECs by modulating ERK signaling. The findings obtained using the zebrafish model provide evidence of the possible risks of chloroxylenol exposure and suggest the importance of more in-depth ecotoxicological studies.

Developmental safety of nirmatrelvir in zebrafish (Danio rerio) embryos.

BACKGROUND: Nirmatrelvir, in combination with ritonavir, is one of the first orally available antiviral treatment for coronavirus disease 2019 (COVID-19). Symptomatic pregnant women are at increased risk for severe illness and complications that can affect the developing baby. No malformations or lower embryo-fetal survival have been observed when nirmatrelvir were administered to pregnant rats and rabbits. Safety evaluation of drugs used for treating COVID-19 also in pregnancy is urgent for public health, then in this study we further investigated nirmatrelvir developmental toxicity using zebrafish as in vivo model. MATERIAL AND METHODS: Using the standardized Fish Embryo Toxicity (FET) test, we first determined the lethal concentration 50 (LC50), exposing embryos from gastrula stage up to 120 hr post fertilization (hpf) and daily recording lethality. Then, we exposed embryos to five doses comprising the human therapeutic one and up to the LC50 (25 µM). Morphology was evaluated at 72 and 120 hpf. RESULTS: Nirmatrelvir did not affect survival rate and did not induce morphological defects up to the human therapeutic dose. Exposure at higher doses (2.4× and 3× the human Cmax ) however resulted in decreased hatching rate, reduced growth, slower heartbeat with pericardial edema, reduction of eye dimension, absence of the swim bladder and disruption of the anterior-posterior axis, with lack of tail detachment, spinal curvature and straight and smaller head. CONCLUSIONS: Our findings in zebrafish embryos add further information about developmental nirmatrelvir safety. Further studies are needed for pharmacological safety assessment of nirmatrelvir exposure during pregnancy.

Developmental toxicity of remdesivir, an anti-COVID-19 drug, is implicated by in vitro assays using morphogenetic embryoid bodies of mouse and human pluripotent stem cells.

BACKGROUND: Remdesivir is an antiviral drug approved for the treatment of COVID-19, whose developmental toxicity remains unclear. More information about the safety of remdesivir is urgently needed for people of childbearing potential, who are affected by the ongoing pandemic. Morphogenetic embryoid bodies (MEBs) are three-dimensional (3D) aggregates of pluripotent stem cells that recapitulate embryonic body patterning in vitro, and have been used as effective embryo models to detect the developmental toxicity of chemical exposures specifically and sensitively. METHODS: MEBs were generated from mouse P19C5 and human H9 pluripotent stem cells, and used to examine the effects of remdesivir. The morphological effects were assessed by analyzing the morphometric parameters of MEBs after exposure to varying concentrations of remdesivir. The molecular impact of remdesivir was evaluated by measuring the transcript levels of developmental regulator genes. RESULTS: The mouse MEB morphogenesis was impaired by remdesivir at 1-8 µM. Remdesivir affected MEBs in a manner dependent on metabolic conversion, and its potency was higher than GS-441524 and GS-621763, presumptive anti-COVID-19 drugs that act similarly to remdesivir. The expressions of developmental regulator genes, particularly those involved in axial and somite patterning, were dysregulated by remdesivir. The early stage of MEB development was more vulnerable to remdesivir exposure than the later stage. The morphogenesis and gene expression profiles of human MEBs were also impaired by remdesivir at 1-8 µM. CONCLUSIONS: Remdesivir impaired mouse and human MEBs at concentrations that are comparable to the therapeutic plasma levels in humans, urging further investigation into the potential impact of remdesivir on developing embryos.

Reproductive and developmental safety of nirmatrelvir (PF-07321332), an oral SARS-CoV-2 Mpro inhibitor in animal models

Nirmatrelvir (PF-07321332;NMV) the antiviral component of PAXLOVID PACK is a potent and selective inhibitor of the SARSCoV- 2 main protease (Mpro), which plays a critical role in viral replication. PAXLOVID PACK, comprised of nirmatrelvir and ritonavir (used as a pharmacokinetic enhancer), is an oral therapy currently in development as a therapeutic option for those infected with SARS-CoV-2 to prevent progression to severe disease, hospitalization, and death. It is also being tested for its potential benefit in the post-exposure prophylactic setting. Given that males and females of reproductive age are included in the intended patient population, we assessed the potential effects of NMV up to the limit dose of 1000 mg/kg/day in ICH guideline embryo-fetal development studies in rats and rabbits, and a fertility and early embryonic development study in rats. There were no effects on male and female fertility or early embryonic development in rats, and no severe manifestations of developmental toxicity in rats or rabbits. The lack of adverse findings reported here in nonclinical species is consistent with the intended therapeutic target of NMV (a virus specific protein not present in mammalian cells), the favorable off-target selectivity profile, and lack of genetic toxicity. All procedures performed on the animals in these studies were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.

In the Swim of Cannabis: Developmental Toxicity and Metabolomic Pathway Alterations of Zebrafish Larvae Exposed to THC for the Assessment of Its Potential Environmental and Human Health Impact.

As the pharmacological properties and therapeutic applications of Cannabis sativa L. pace with the upsurge of interest of the scientific community in harnessing its constituent phytocannabinoids, illicit use may raise serious health issues. Tetrahydrocannabinol (THC) is one of the most well-known phytoactive constituents of cannabis and continues to garner scientific and public attention not only because of its pharmacological value but also because over-the-counter products of THC and prescription medications are becoming increasingly available from pharmacies, dispensaries, Internet, local retail stores, or by illicit means. Hence, a multidimensional approach was employed to examine the impact of THC on zebrafish larvae. The acute toxicity, expressed as LC50, was 1.54 mg/L. Adverse effects were observed on the phenotype, such as tail bending, pericardial edema, etc., even at concentrations lower than LC50, and fundamental functions of larvae (e.g., heart rate and cardiac contractility, and rhythm) were significantly affected. Behavioral changes were noticed, which were reflected in locomotor activity and sensitivity to light/dark changes. Finally, an untargeted metabolomic study was carried out to shed light on the metabolic alterations that occurred, providing substantiating evidence of the observed phenotype alterations. Overall, the potentially detrimental effects of THC on a vertebrate model are depicted.

Investigation of developmental toxicity of favipiravir on fetal bone and embryonic development.

BACKGROUND: Favipiravir is one of the essential antiviral drugs used for the treatment of coronavirus disease (COVID-19) in some countries. However, there is not enough information about used, especially in pregnancy. Therefore, in this study, it was aimed to determine the developmental toxicity of favipiravir on fetal bone development and embryonic development. METHODS: In this study, 16 pregnant wistar albino rats were used. The rats were divided into four groups: Control (saline) and Group A (50 mg/kg × 5 days), Group B (50 mg/kg × 1 days + 20 mg/kg × 4 days), Group C (20 mg/kg × 5 days). Solutions were administered to the rats by oral gavage from the 10th to 14th days of pregnancy, twice a day. The skeletal system development of fetuses was examined with double skeletal staining and immunohistochemical staining methods. RESULTS: A total of 72 fetuses from pregnant rats, 18 in each group, were included in the study. As a result, depending on favipiravir dose increase, in experimental groups, it was determined that the statistically significant decrease on the ossification rates of anterior and posterior extremity bones, and length and weight of fetuses. CONCLUSION: Exposure to favipiravir during pregnancy impairs bone metabolism and bone formation-resorption stages and may cause developmental delay.

Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae).

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 µg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences.

Lack of effects on female fertility and prenatal and postnatal offspring development in rats with BNT162b2, a mRNA-based COVID-19 vaccine.

BNT162b2 is a vaccine developed to prevent coronavirus disease 2019 (COVID-19). BNT162b2 is a lipid nanoparticle formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein locked in its prefusion conformation. A developmental and reproductive toxicity study was conducted in rats according to international regulatory guidelines. The full human BNT162b2 dose of 30 µg mRNA/dose (>300 times the human dose on a mg/kg basis) was administered intramuscularly to 44 female rats 21 and 14 days prior to mating and on gestation days 9 and 20. Half of the rats were subject to cesarean section and full fetal examination at the end of gestation, and the other half were allowed to deliver and were monitored to the end of lactation. A robust neutralizing antibody response was confirmed prior to mating and at the end of gestation and lactation. The presence of neutralizing antibodies was also confirmed in fetuses and offspring. Nonadverse effects, related to the local injection site reaction, were noted in dams as expected from other animal studies and consistent with observations in humans. There were no effects of BNT162b2 on female mating performance, fertility, or any ovarian or uterine parameters nor on embryo-fetal or postnatal survival, growth, physical development or neurofunctional development in the offspring through the end of lactation. Together with the safety profile in nonpregnant people, this ICH-compliant nonclinical safety data supports study of BNT162b2 in women of childbearing potential and pregnant and lactating women.

Vertical transmission and kidney damage in newborns whose mothers had coronavirus disease 2019 during pregnancy.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) has become a worldwide pandemic. However, the hazard to newborns in pregnancy remains controversial. The aim of this study was to investigate the vertical transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from mother to child and developmental toxicity in the fetus. METHODS: All clinical information was recorded on 22 neonates born to mothers with confirmed COVID-19 pneumonia in Tongji Hospital. RESULTS: The average birth weight of the 22 newborns (16 males and 6 females) was 2980 g, and the mean gestational week was 37W+3. The birth weight of three babies was <2500 g, and the gestational week of all three low-birth-weight neonates was less than 36W. Three newborns had minor lesions of infection in the lungs as shown by computed tomography (CT) scans. Furthermore, three newborns had elevated SARS-CoV-2-related immunoglobin M (IgM) antibodies, and 11 newborns (52.4%) had positive immunoglobin G (IgG) antibodies. Notably, both cystatin C and ß2-microglobulin were increased in all newborns. Five of the 21 tested newborns had leukocytosis, and 11 had increased neutrophil levels. In addition, the aspartate aminotransferase of 18 newborns and the γ-glutamyl transpeptidase of 19 newborns were increased. Total bilirubin was elevated in all newborns and serum albumin was reduced in 20 of 22 newborns. CONCLUSIONS: This study was the first to discover that COVID-19 infection in the third trimester of pregnancy could cause fetal kidney developmental injury, as indicated by increased cystatin C and ß2-microglobulin in all neonates. Furthermore, there is the possibility of maternal-fetal transmission of SARS-CoV-2.