Perfluoroalkyl acids (PFAAs) have been widely used since 1950s. The long chained-PFAAs, such as perfluorooctanoic acid (PFOA) are persistent and bio-accumulative, and are detected in humans. PFOA, which is a peroxisome proliferator activated receptor (PPAR) α agonist, has been suggested to be a carcinogen in epidemiological and animal studies. In some studies PFOA is shown to be non-mutagenic in Ames and micronucleus tests, but in other studies it caused oxidative DNA damage and micronucleus formation. However, there has been no report that has examined whether PFOA-induced genotoxicity is mediated by PPARα. In order to relate genotoxicity of PFAAs to PPARα, we conducted two kinds of comet assays (cellular and acellular), a micronucleus (MN) test, and a TK mutation assay with and without PPARαantagonists by using human lymphoblastoid cells. PFAAs at 125-1000 µg/mL showed positive responses in the cellular comet assay but not in the MN test and TK mutation assay. A PPARα antagonist GW6471 (2 µg/mL) only partly reduced PFOA-induced DNA damage (in the cellular comet assay), but abolished PFOA-induced intracellular ROS formation. PFAAs with 8-12 carbons also showed positive responses in the acellular comet assay where there is no cellular function such as PPAR. Therefore, PFOA-induced DNA damage was partly related to the oxidative stress via PPARα, without manifestation of chromosome aberration and point mutation in this cell line.
Carbon tetrachloride (CCl4) is a well-known hepatotoxic chemical. Exposure to CCl4 produces free radicals, which induce oxidative stress and cause hepatic injury. We demonstrated previously that pretreatment with zinc (Zn), which induces metallothionein (MT) expression, prevents CCl4-induced lethal toxicity in a dose-dependent manner. While MT has been suggested as a possible hepatoprotective protein, its mechanism of protection remains unknown. In the current study, we evaluated the protective mechanism of MT, an endogenous scavenger of free radicals, against CCl4-induced toxicity through subcutaneous administration of 50 mg/kg Zn (as ZnSO4) once daily for three consecutive days, prior to a single intraperitoneal injection of 4 g/kg CCl4 in male ddY mice. Our results showed that Zn pretreatment significantly decreased aspartate aminotransferase and total cholesterol levels, 6-hr after CCl4 injection, as well as lipid peroxidation. Moreover, CCl4-induced hepatic calcium level was downregulated by pretreatment with Zn while Zn-induced MT expression decreased by more than 500 µg/g liver (43%) in the Zn + CCl4-treated group, implying that MT was consumed by CCl4-induced free radicals. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute hepatotoxicity, presumably by inducing the expression of free radical-scavenging MT.
The Bhas 42 cell transformation assay is a sensitive and effective mutation assay, and it has been widely used to detect and evaluate potential carcinogenic risks, including initiation assay and promotion assay. In this transformation assay, the number of transformed foci is the only criterion. However, it is nonquantitative because the shape is irregular. Nevertheless, a high-throughput Bhas 42 cell transformation assay was developed, which includes the usage of hydrogen peroxide in 96-well plates. In this way, transformed foci are quantitatively analysed in proportion to the optical density value (OD value). In this study, the Bhas 42 cell transformation assay was established using the criterion of the OD value. As a result, 3-methylcholanthrene (3-MCA) presented strong tumour initiation activity, whereas the 12-O-tetradecanoylphorbol-13-acetate (TPA) showed strong tumour promotion activity. Lowdaphne Stringbush Flower and Leaf (LSFL, also named as Wikstroemia chamaedaphne (Bunge) Meissn) and Rhubarb (Rheum palmatum L.) showed strong tumour promotion activity, but the tumour promotion activity of Lilac Daphne Flower Bud (LDFB, also named as Daphne genkwa Siebold & Zucc) was equivocal. Surprisingly, the OD value displayed a strong correlation with the number of transformed foci. In conclusion, the high-throughput Bhas 42 cell transformation assay was feasible.
The Pig-a gene is well-known to encode a key enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI), which attaches CD molecules (Cluster differentiation), such as CD55 and CD59, to red blood cells (RBCs) membranes. In this study, the blood was marked with the special antigen CD45-PE (Phycoerythrin) to separate the erythrocytes from the leukocytes, then the Pig-a mutant frequency (MF) of RBCs could be investigated without pivotal antigen CD59-FITC (Fluorescein isothiocyanate), and the optimal ENU concentration was determined to be 100 mg/kg/day. The optimal cell number to count and the stability of specimens were determined. At last, the in vivo Pig-a gene mutation assay was utilized to detect the potential genotoxicity of Cis-Dichlorodiammineplatinum (DP), Procarbazine Hydrochloride (PH), and Triptolide (TP). The results indicated that the Pig-a gene mutation obviously occurred in the PH treatment group. In the DP treatment group, an irregular shape with a slightly serrated border in erythrocytes was observed in the blood smear. However, no obvious Pig-a gene mutations were detected in the DP treatment and TP treatment groups under the experimental conditions. In conclusion, this primary in vivo Pig-a gene mutation assay will provide an easy protocol to use in screening potential genotoxic compounds.
In hospitals and pharmacies, many kinds of pharmaceutical tablets are frequently crushed to powder in order to facilitate administration. However, there is concern about the influence of this process on the health of pharmacy workers. In this study, we conducted model experiments to estimate the potential exposure of pharmacy workers to pharmaceutical particles during tablet crushing and transfer of powder. Tablets were crushed in a tablet mill. Particulates released into the air during and after milling and transfer to a mortar were counted with a dust counter, and collected on the filter of an air sampler. Amounts of pharmaceutical active ingredients collected on the filter were determined by high-performance liquid chromatography (HPLC). During tablet crushing, particulates were released into the air. We found that the particle concentration in air was highest during transfer of the powder from the tablet mill to the mortar. The amount of active ingredient collected on the filter of the air sampler was significantly higher in the case of Loxonin, as compared with Neurovitan. Although conditions under which tablets are crushed are likely to vary greatly in practice, our results and calculations at least indicate that unmasked workers might routinely inhale microgram levels of active ingredients during tablet crushing and transfer of the resulting powder. Our results should be helpful in designing appropriate protective measures and in developing professional guidelines to minimize occupational exposure of pharmacy workers to drugs.
Female C57BL mice were exposed to a low level of mercury vapor (Hg0, 0.096 mg/m3) and was given the solution containing 5-ppm methylmercury (MeHg) during the growth period to examine the influence on the neurobehavioral function after birth. Exposure period was 4 weeks at 3 to 7 weeks of age. At 10 weeks of age, three behavioral tests were conducted; open field (OPF) test, passive avoidance response (PA) test, and eight-arm radial maze (RM) test. To evaluate the influence of aging, additional behavioral tests were performed at 79 weeks of age. With respect to the results of the three behavioral tests conducted at 10 to 14 weeks of age, there were no significant differences between the Hg0/MeHg/Hg0+MeHg and control groups. Furthermore, there were also no significant differences between each exposure and control group on behavioral tests performed at 79 to 83 weeks of age after the completion of mercury exposure. The concentration of mercury in the brain after the completion of exposure was the highest in the Hg0+MeHg group, followed by the MeHg and Hg0 groups. The values in the Hg0+MeHg and MeHg groups were ≤ 3.0 µg/g. The value in the Hg0 group was ≤ 1.0 µg/g. There were no differences in the brain concentration of mercury after 1 year between the Hg0/MeHg and control groups. However, in the Hg0+MeHg group, it was significantly higher than in the control group, suggesting that the disappearance of mercury in the brain is delayed in comparison with the exposure to Hg0 or MeHg alone. These results showed that there was no influence of low-level Hg0+MeHg exposure during the growth period on neurobehavioral manifestation. However, the disappearance of mercury in the brain was delayed in comparison with the exposure to Hg0 or MeHg alone.