Toxicological Assessment of Bromochlorophene: Single and Repeated-Dose 28-Day Oral Toxicity, Genotoxicity, and Dermal Application in Sprague-Dawley Rats.

Bromochlorophene (BCP) has shown good properties in sterilization and antibacterial activity and is widely used as a household chemical. We evaluated the genotoxicity, single and repeated-dose 28-day oral toxicity, and dermal application of a BCP suspension in Sprague-Dawley (SD) rats. For the single-dose toxicity study, a dose of 25-1,000 mg per kg of bodyweight (mg/kg b.w.) of BCP was given once orally to SD rats. Mortality and clinical signs were observed and recorded for the first 30 min after treatment, at 4 h post-administration, and then at least once daily for 14 days after administration. For the repeated-dose 28-day toxicity study, the high dose was set at 1,000 mg/kg b.w. and the middle, middle-low, and low dose were set to 500, 250, and 125 mg/kg, respectively. Hematology and biochemistry parameters were examined. Gross pathologic and histopathologic examinations were performed on selected tissues from all animals. A bacterial reverse mutation assay, in vitro chromosomal aberration assay, and in vivo micronucleus assay were performed to assess genotoxicity-dermal application exposure assessment of BCP in rats. A high oral approximate lethal dose (ALD) of 1,000 mg/kg was observed in the single-dose toxicity test. During the repeated-dose 28-day time period, most animal deaths after administration occurred during the first 3 weeks. The 1,000 mg/kg b.w. oral dose caused the death of six male rats (6/7) and four female rats (4/7). At 500 mg/kg b.w., the female rats showed mortality (1/7). For the biochemistry assays, cholesterol was increased significantly compared to vehicle in both sexes in the 250 and 500 mg/kg groups. Histopathological changes with treatment-related findings were observed in the pancreas in female rats treated with a high dose of BCP compared with the vehicle group. BCP showed no genotoxic effect. These data suggested that the ALD of BCP, estimated as a non-genotoxic substance, was over 1,000 mg/kg b.w. in the single-dose toxicity study, and the NOAEL of BCP was considered to be 250 mg/kg b.w. for male and female rats after repeated oral administration for 28 days under the present study conditions.

Corrigendum: Copper and Cobalt Ions Released From Metal Oxide Nanoparticles Trigger Skin Sensitization.

[This corrects the article DOI: 10.3389/fphar.2021.627781.].

Skin Sensitization Evaluation of Carbon-Based Graphene Nanoplatelets.

Graphene nanoplatelets (GNPs) are one of the major types of carbon based nanomaterials that have different industrial and biomedical applications. There is a risk of exposure to GNP material in individuals involved in their large-scale production and in individuals who use products containing GNPs. Determining the exact toxicity of GNP nanomaterials is a very important agenda. This research aimed to evaluate the skin sensitization potentials induced by GNPs using two types of alternative to animal testing. We analyzed the physicochemical characteristics of the test material by selecting a graphene nanomaterial with a nano-size on one side. Thereafter, we evaluated the skin sensitization effect using an in vitro and an in vivo alternative test method, respectively. As a result, we found that GNPs do not induce skin sensitization. In addition, it was observed that the administration of GNPs did not induce cytotoxicity and skin toxicity. This is the first report of skin sensitization as a result of GNPs obtained using alternative test methods. These results suggest that GNP materials do not cause skin sensitization, and these assays may be useful in evaluating the skin sensitization of some nanomaterials.

Copper and Cobalt Ions Released from Metal Oxide Nanoparticles Trigger Skin Sensitization.

Human skins are exposed to nanomaterials in everyday life from various sources such as nanomaterial-containing cosmetics, air pollutions, and industrial nanomaterials. Nanomaterials comprising metal haptens raises concerns about the skin sensitization to nanomaterials. In this study, we evaluated the skin sensitization of nanomaterials comparing metal haptens in vivo and in vitro. We selected five metal oxide NPs, containing copper oxide, cobalt monoxide, cobalt oxide, nickel oxide, or titanium oxide, and two types of metal chlorides (CoCl2 and CuCl2), to compare the skin sensitization abilities between NPs and the constituent metals. The materials were applied to KeratinoSensTM cells for imitated skin-environment setting, and luciferase induction and cytotoxicity were evaluated at 48 h post-incubation. In addition, the response of metal oxide NPs was confirmed in lymph node of BALB/C mice via an in vivo method. The results showed that CuO and CoO NPs induce a similar pattern of positive luciferase induction and cytotoxicity compared to the respective metal chlorides; Co3O4, NiO, and TiO2 induced no such response. Collectively, the results implied fast-dissolving metal oxide (CuO and CoO) NPs release their metal ion, inducing skin sensitization. However, further investigations are required to elucidate the mechanism underlying NP-induced skin sensitization. Based on ion chelation data, metal ion release was confirmed as the major "factor" for skin sensitization.

Evaluation of the Skin Sensitization Potential of Carbon Nanotubes Using Alternative In Vitro and In Vivo Assays.

Carbon nanotubes (CNTs) are one of the major types of nanomaterials that have various industrial and biomedical applications. However, there is a risk of accidental exposure to CNTs in individuals involved in their large-scale production and in individuals who use products containing CNTs. This study aimed to evaluate the skin sensitization induced by CNTs using two alternative tests. We selected single-wall carbon nanotubes and multi-walled carbon nanotubes for this study. First, the physiochemical properties of the CNTs were measured, including the morphology, size, and zeta potential, under various conditions. Thereafter, we assessed the sensitization potential of the CNTs using the ARE-Nrf2 Luciferase KeratinoSens™ assay, an in vitro alternative test method. In addition, the CNTs were evaluated for their skin sensitization potential using the LLNA: BrdU-FCM in vivo alternative test method. In this study, we report for the first time the sensitization results of CNTs using the KeratinoSens™ and LLNA: BrdU-FCM test methods in this study. This study found that both CNTs do not induce skin sensitization. These results suggest that the KeratinoSens™ and LLNA: BrdU-FCM assay may be useful as alternative assays for evaluating the potential of some nanomaterials that can induce skin sensitization.

Effect on the H19 gene methylation of sperm and organs of offspring after chlorpyrifos-methyl exposure during organogenesis period.

To elucidate the effect on the H19 gene methylation of sperm and organs in offspring by chlorpyrifos-methyl (CPM) exposure during organogenesis period, CPM was administered at doses of 4 (CPM4), 20 (CPM20), and 100 (CPM100) mg/kg bw/day from 7 days post coitum (d.p.c.) to 17 d.p.c. after mating CAST/Ei (♂) and B6 (♀). Anogenital distance (AGD) was measured at postnatal day (PND) 21. Clinical signs, body weights, feed and water consumption, organs weights, serum hormone values, and H19 methylation level of organ and sperm were measured at PND63. Body weights were significantly lower than control until PND6. AGD was significantly decreased in the CPM100 group in males and increased in the CPM20 group in females. The absolute weights of the thymus and epididymis were significantly increased for males in all of CPM treatment groups. In the CPM20 group, absolute weights of liver, kidney, heart, lung, spleen, prostate gland, and testes were significantly increased. Testosterone concentrations in serum were significantly increased by CPM treatment in males. H19 methylation level of liver and thymus showed decreased pattern in a dose-dependent manner in males. The levels of H19 methylation in sperm were 73.76 ± 7.16% (Control), 57.84 ± 12.94% (CPM4), 64.24 ± 3.79% (CPM20), and 64.24 ± 3.79% (CPM100). Conclusively, CPM exposure during organogenesis period can disrupt H19 methylation in sperm, liver, and thymus and disturb the early development of offspring.

Exposure of pregnant mice to chlorpyrifos-methyl alters embryonic H19 gene methylation patterns.

The aim of this study was to identify whether chlorpyrifos methyl (CPM) exposure during pregnancy leads to changes in the methylation patterns of H19 gene. CPM 4, 20, 100 mg/kg bw/day was administered to 4 pregnant mice per group between 7 and 12 days post coitum (d.p.c.). Pregnant mice were killed at 13 d.p.c. The genomic methylation in primordial germ cells (PGCs) and fetal organs (the liver, intestine, and placenta) was examined. Four polymorphism sites in the H19 alleles of maternal (C57BL/6J) and paternal (CAST/Ei) alleles were identified at nucleotide position 1407, 1485, 1566, and 1654. The methylation patterns of 17 CpG sites were analyzed. The methylation level in male and female PGCs was not altered by CPM treatment in the maternal allele H19. The methylation level of the paternal H19 allele was altered in only male PGCs in response to the CPM treatment. The methylation level at a binding site for the transcriptional regulator CTCF2 was higher than that at the CTCF1 binding site in all CPM-treated groups. In the placenta, the aggregate methylation level of H19 was 56.89%in control group. But, those levels were ranged from 47.7% to 49.89% after treatment with increasing doses of CPM. H19 gene from the liver and intestine of 13 d.p.c. fetuses treated with CPM was hypomethylated as compared with controls, although H19 mRNA expression was unaltered. In the placenta, H19 expression was slightly increased in the CPM-treated group, although not significantly. IGF2 expression levels were not significantly changed in the placenta. In conclusion, CPM exposure during pregnancy alters the methylation status of the H19 gene in PGCs and embryonic tissues. We infer that these alterations are likely related to changes in DNA demethylase activity.

Toxic effects of methylmercury, arsanilic acid and danofloxacin on the differentiation of mouse embryonic stem cells into neural cells.

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABAA-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABAA-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 µM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.

Effects of oral deoxynivalenol exposure on immune-related parameters in lymphoid organs and serum of mice vaccinated with porcine parvovirus vaccine.

Mice were exposed to deoxynivalenol (DON) via drinking water at a concentration of 2 mg/L for 36 days. On day 8 of treatment, inactivated porcine parvovirus vaccine (PPV) was injected intraperitoneally. The relative and absolute weight of the spleen was significantly decreased in the DON-treated group (DON). Antibody titers to parvovirus in serum were 47.9 ± 2.4 in the vaccination group (Vac), but 15.2 ± 6.5 in the group treated with DON and vaccine (DON + Vac). The IgA and IgG was not different in the DON, Vac an,d DON + Vac groups. IgM was significantly lower only in the DON + Vac group. However IgE was significantly increased in the Vac and DON + Vac group, but no change was observed between the Vac and DON + Vac groups. The concentrations of IL-2, IL-4, GM-CSF, MCP-1 and Rantes in serum, and IL-1α in mesenteric lymph node and MIP-1ß in spleen were significantly increased by DON treatment compared to control. The concentrations of IL-2, IL-5, IL-6, IL-9, IL-12, IL-13 and Rantes in thymus, of IL-2 in spleen, and of IL-1α, IL-1ß, IL-3, IL-5, IL-10, IL-17, G-CSF, GM-CSF and MCP-1 in mesenteric lymph nodes were significantly decreased in mice compared to those in the Vac group, while concentrations of IL-1α, IL-2, IL-9, IL-13,G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and TNF-α were significantly increased in serum compared to the Vac group. In conclusion, the results presented here indicate that exposure to DON at 2.0 mg/L via drinking water can disrupt the immune response in vaccinated mice by modulating cytokines and chemokines involved in their immune response to infectious disease.

Evaluation of hepatotoxicity of chemicals using hepatic progenitor and hepatocyte-like cells derived from mouse embryonic stem cells: effect of chemicals on ESC-derived hepatocyte differentiation.

Embryonic stem cell testing is an alternative model system to assess drug and chemical toxicities because of its similar developmental characteristics with in vivo embryogenesis and organogenesis. This study evaluated the toxicity of chemicals at specific developmental stages of mouse embryonic stem cell (ESC)-derived hepatic differentiation; hepatic progenitor cells (HPCs), and hepatocyte-like cells (HCs). The toxic effects of carbon tetrachloride (CCl(4)), 5-fluorouracil (5-FU), and arsanilic acid (Ars) were evaluated by measuring the expressions of Cytokeratin (CK18) and GATA binding protein 4 (GATA-4) and the activities of aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) during the hepatic differentiation process. Non-toxic doses of three chemicals at a range of 25 to 500 µM for CCl(4), 12.5 to 800 nM for 5-FU and 6.25 to 400 mM for Ars were treated. In the CCl(4)-treated group, significant decreases (P < 0.05) of the marker expression were observed by more than 300 µM from day 10 in CK18 and by more than 400 µM of CCl(4) from day 22 in GATA-4, respectively. However, both markers were decreased (P < 0.01) by treatments of all doses at day 40. In the 5-FU-treated group, the expressions of two proteins were not affected by any of the doses at day 10 and 22, whereas the GATA-4 expression was decreased (P < 0.05) by more than 400 nM of 5-FU at days 28 and 40. In the Ars-treated group, the CK18 expression was inhibited (P < 0.05) by more than 100 mM of Ars at day 22 but showed a tendency to recover. Although the GATA-4 was inhibited by all doses at day 22, the inhibition of GATA-4 recovered at days 28 and 40. ALP activities of three chemicals were significantly increased (P < 0.05) by a dose-dependent manner. The activities of AST and LDH were prone to be increased by more than 300 µM of CCl(4,) but not affected by all doses of 5-FU except for 800 nM of 5-FU in AST activities. In the Ars, the enzyme activities were significantly increased (P < 0.05) by more than 50 µM of Ars in AST and more than 6.25 µM of Ars in LDH. The present results indicate that CCl(4) has a more toxic effect on HCs, whereas Ars is more toxic to HPCs. Additionally, in vitro alternative testing using ESC-derived HPCs and HCs could provide useful information on chemical toxicity during the hepatic differentiation process and could be a useful model system for assessing chemical hepatotoxicity.

Zearalenone affects immune-related parameters in lymphoid organs and serum of rats vaccinated with porcine parvovirus vaccine.

Rats were administered zearalenone (ZEA) via gavage at dosages of 0, 1, 5, and 30 mg/kg for 36 days. On treatment day 8, inactivated porcine parvovirus vaccine (Vac) was injected intraperitoneally. Antibody production against porcine parvovirus was then measured as a function of ZEA treatment. Compared to the vaccine alone, ZEA treatment, with or without Vac, decreased the serum level of IgG. The level of IgM decreased in all ZEA groups at day 22, but the decrease was sustained only in the medium-dose ZEA group at day 36. The level of IgA was unchanged in the Vac only and ZEA groups at day 22, but was decreased in the 5 mg/kg ZEA plus Vac group compared to the Vac only group at day 36. The level of IgE was decreased by all doses of ZEA at day 22, but was unaffected in ZEA plus Vac groups compared to the Vac only group. The levels of IL-1 in the thymus and spleen; INF-γ in serum; IL-2, IL-6, and IL-10 in the thymus; and IL-10 and IFN-γ in the spleen decreased after ZEA administration. Furthermore, the levels of IL-1ß in the spleen and mesenteric lymph node, IL-1ß in the thymus, IL-2 in the thymus and spleen, IL-6 in the thymus, IL-10 and IFN-γ in the spleen, and GM-CSF and TNF-α in the thymus decreased after vaccination in rats exposed to ZEA. In conclusion, these results suggest that ZEA exposure via drinking water can cause an immunosuppressive effect by decreasing immunoglobulins in serum and cytokines in lymphoid organs.