The Journal of Toxicological Sciences Vol. 42(2017) No. 3
June

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor which is abundant in the liver, intestine, and kidney. FXR is a pivotal factor in cholesterol/bile acid homeostasis but is involved in the growth of hepatocellular carcinoma cells. In the present study, we investigated whether FXR is also involved in the growth of renal adenocarcinoma cells. The cell growth of renal adenocarcinoma cell line ACHN was inhibited by FXR knockdown and stimulated by FXR ligand, while that of a normal renal cell-derived cell line, HK-2, was not affected. The carcinoma-specific stimulation of cell growth by FXR was found to arise from down-regulation of p53 and p21/Cip1 mRNA expression. Our study showed that FXR stimulates proliferation of renal adenocarcinoma cells and that FXR knockdown is useful for growth suppression of renal adenocarcinoma without cytotoxicity to normal renal cells.

Herein, we report on the joint toxicity of four fluoroquinolones and two tetracyclines (β-diketone antibiotics-DKAs) to zebrafish based on a series of toxicological endpoints and histopathological observations. A positive dose-dependence was observed in DKA-exposure groups with a 72-hpf EC₅₀ of 130.3 mg/L for hatching rate, 120-hpf LC₅₀ of 149.8 mg/L, and 120-hpf EC₅₀ of 135.1 mg/L for malformation rate. When zebrafish at 60 dpf were exposed to a series of DKA concentrations (45, 60 and 90 mg/L) for 7, 14 and 21 days, creatine kinase and AChE activities were significantly induced, and intracellular malondialdehyde increased in all treatments except for the 45 mg/L treatment. The transcription levels of AHRRa from livers were significantly (p < 0.05) up-regulated in all treatments after two months of DKA exposure. CKma expression from skeletal muscle was significantly down-regulated in the 90 mg/L treatment. A remarkable down-regulation of CYP3A65 was observed in the 60 mg/L treatment. DKA exposure resulted in severe tissue damage including mitochondria swelling, reduction of mitochondrial cristae, deepening of mitochondrial cristae bands, and decreasing and even disappearance of the rough endoplasmic reticulum. Total sperm motility was decreased by ca. 30% due to DKA exposure. These results provide important information for toxicity and health risks due to mixed DKA exposure in aquatic environments.

Bisphenol-A (BPA) is an estrogenic endocrine disruptor mostly used for the production of polycarbonate plastics and epoxy resins. Recently we have reported that perinatal BPA exposure impaired spatial memory through upregulation of synaptic proteins Neurexin1 and Neuroligin3 in male mice. As epigenetic mechanism is a key regulator of memory, we hypothesized that BPA might influence memory through epigenetic regulation of gene expression. Here we provide evidence that perinatal exposure to BPA decreased 5-mC DNA but increased histone H3 acetylation in cerebral cortex and hippocampus of postnatal 3 and 8 weeks male mice. BPA exposure also increased mRNA levels of DNMT1 and DNMT3a in cerebral cortex of 3 and 8 weeks; whereas in hippocampus DNMT1 mRNA increased in 3 weeks but decreased in 8 weeks and DNMT3a showed no change. Further, HDAC2 mRNA and protein increased in cerebral cortex of both ages and in hippocampus it increased in 3 weeks but decreased in 8 weeks. Altogether, our results demonstrate that the perinatal BPA exposure induces epigenetic changes that possibly underlie the enduring effect of BPA on brain function and behavior.

S-1 is an anticancer agent that consists of tegafur, gimeracil, and oteracil potassium at a molar ratio of 1:0.4:1. S-1 is used to treat metastatic and resectable gastric cancer. However, the extensive use of S-1 in clinical practice results in watery eyes, a serious clinical problem, which worsens patients’ quality of life. Although repeated instillation of artificial tears is recommended, therapy or prophylaxis against S-1-induced ocular toxicity has not been established. In the present study, we evaluated the alleviating effects of repeated artificial tear instillation on S-1-induced ocular toxicity in dogs. Ten beagle dogs (5 males and 5 females) were orally administered 3 mg/kg/day of S-1 for up to 21 days. Five drops of artificial tears were instilled to the left eye, eight times daily, within 6 hr after S-1 administration. The mean cornea staining score tended to be low in the left eye with repeated artificial tear instillation. In 4 out of 10 dogs, the corneal staining score of the left eye was more than 2-fold lower than that of the right eye. The incidence of dogs indicating normal tear drainage increased and stenosed tear drainage decreased by repeated artificial tear instillation. In conclusion, we demonstrated that artificial tear instillation can alleviate corneal surface damage induced by S-1 in dogs.

To examine the kinetics of low doses of perfluoro compounds (PFCs), we administered perfluorohexanoic acid (C6A), perfluorooctanoic acid (C8A), perfluorononanoic acid (C9A) and perfluorooctane sulfonate (C8S) with a single oral dose (50-100 μg/kg BW), and in drinking water at 1, 5, and 25 μg/L for one and three months to male rats; and examined the distribution in the brain, heart, liver, spleen, kidney, whole blood and serum. C6A was very rapidly absorbed, distributed and eliminated from the tissues with nearly the same tissue t_1/2 of 2-3 hr. Considering serum Vd, and the tissue delivery, C6A was mainly in the serum with the lowest delivery to the brain; and no tissue accumulation was observed in the chronic studies as estimated from the single dose study. For the other PFCs, the body seemed to be an assortment of independent one-compartments with a longer elimination t_1/2 for the liver than the serum. The concentration ratio of liver/serum increased gradually from C₀ to a steady state. The high binding capacity of plasma protein may be the reason for the unusual kinetics, with only a very small fraction of free PFCs moving gradually to the liver. Although the tissue specific distribution was time dependent and different among the PFCs, the Vd and k_e of each tissue were constant throughout the study. The possibility of extremely high C6A accumulation in the human brain and liver was suggested, by comparing the steady state tissue concentration of this study with the human data reported by Pérez et al. (2013).

Mono-butyl phthalate (MBP) has reproductive toxicity but the related mechanisms have not been fully elucidated in vivo. We exposed male Balb/c mice to MBP by gavage at doses of 0, 25, 50, 100, 200 mg/kg for 14 days, and then evaluated the testicular alterations at the histological and molecular levels. MBP reduced mouse sperm count along with sperm malformation and seminiferous tubule degeneration in a dose-dependent manner. MBP dosed at 200 mg/kg significantly increased reactive oxygen species and malondialdehyde content in mouse testes. High doses of MBP (200 mg/kg) also significantly reduced mRNA expressions of testis growth and function related genes (Sox9 and Dazl). Our findings suggest that oxidative stress and down-regulated expression of Sox9 and Dazl may play important roles in MBP-induced testis injury.

In a cerebrum damaged by methylmercury, where neuropathological lesions tend to localize along deep sulci and fissures, edematous changes in white matter have been proposed as the cause of such localization. Since hyaluronan has a high water-retention capability and can contribute to the progression of edematous changes, we hypothesize that methylmercury increases hyaluronan in brain microvascular cells. Our experimental results indicate that methylmercury induces the expression of hyaluronan in cultured human microvascular endothelial cells and pericytes through the induction of expressed UDP-glucose dehydrogenase and hyaluronan synthase 2, respectively. After exposure to methylmercury, hyaluronan largely accumulates in perivascular space, where it contributes to the progression of edematous changes.

Drug abusers most often smoke ‘herbal incense’ as a cigarette or inhale it using a smoking tool. Smoking may cause pyrolysis of the drug and produce decomposed products of which biological effect has never been investigated. The synthetic cannabinoid UR-144 is known to undergo thermal degradation, giving a ring-opened isomer, so-called UR-144 degradant. The present study demonstrates by using UR-144 as a model drug that the smoke of burned UR-144 contains the UR-144 degradant. The UR-144 degradant showed approximately four fold higher agonist activity to human CB₁ receptor and augmented hypothermic and akinetic actions in mice compared to UR-144. These results indicate that smoking behavior may increase psychological actions of the certain synthetic cannabinoids.

Evidence has been presented for auto-induced human cytochrome P450 3A enzyme involvement in the teratogenicity and clinical outcome of thalidomide due to oxidation to 5-hydroxythalidomide and subsequent metabolic activation in livers. In this study, more relevant human placenta preparations and placental BeWo cells showed low but detectable P450 3A4/5 mRNA expression and drug oxidation activities. Human placental microsomal fractions from three subjects showed detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. Human placental BeWo cells, cultured in the recommended media, also indicated detectable midazolam 1´- and 4-hydroxylation and thalidomide 5-hydroxylation activities. To reduce any masking effects by endogenous hormones used in the recommended media, induction of P450 3A4/5 mRNA and oxidation activities were measured in placental BeWo cells cultured with a modified medium containing 5% charcoal-stripped fetal bovine serum. Thalidomide significantly induced P450 3A4/5, 2B6, and pregnane X receptor (PXR) mRNA levels 2 to 3-fold, but rifampicin only enhanced P450 3A5 and PXR mRNA under the modified media conditions. Under these modified conditions, thalidomide also significantly induced midazolam 1´-hydroxylation and thalidomide 5-hydroxylaion activities 3-fold but not bupropion hydroxylation activity. Taken together, activation of thalidomide to 5-hydroxythalidomide with autoinduction of P450 3A enzymes in human placentas, as well as livers, is suggested in vivo.

Drug-induced liver injury is a major cause of safety-related drug-marketing withdrawals. Several drugs have been reported to disrupt mitochondrial function, resulting in hepatotoxicity. The development of a simple and effective in vitro assay to identify the potential for mitochondrial toxicity is thus desired to minimize the risk of causing hepatotoxicity and subsequent drug withdrawal. An in vitro test method called the "glucose-galactose" assay is often used in drug development but requires prior-culture of cells over several passages for mitochondrial adaptation, thereby restricting use of the assay. Here, we report a rapid version of this method with the same predictability as the original method. We found that replacing the glucose in the medium with galactose resulted in HepG2 cells immediately shifting their energy metabolism from glycolysis to oxidative phosphorylation due to drastic energy starvation; in addition, the intracellular concentration of ATP was reduced by mitotoxicants when glucose in the medium was replaced with galactose. Using our proposed rapid method, mitochondrial dysfunction in HepG2 cells can be evaluated by drug exposure for one hour without a pre-culture step. This rapid assay for mitochondrial toxicity may be more suitable for high-throughput screening than the original method at an early stage of drug development.

Titanium dioxide nanoparticles (TiNPs) present toxicity in organs such as the liver, lung, and intestine. The testis has also been reported as a target organ of TiNPs. We recently reported that TiNPs had no genotoxic effect in the liver and bone marrow, while showing clear testicular dysfunction. In this paper, therefore, we systematically compared the sensitivity of hepatic function using biochemical markers and testicular function against TiNPs. Male C57BL/6J mice were injected intravenously with TiNPs (Aeroxide-P25, at doses of 0.1, 1, 2, and 10 mg/kg body weight) once per week for 4 consecutive weeks. Mice were sacrificed three days after the last injection. Body weights, liver weights, and testicular-related organ weights were not found to be changed by TiNP treatment. Moreover, TiNPs caused no hepatic damage, as evaluated by alanine aminotransferase and aspartate aminotransferase indexes. The testicular function, however, was clearly impaired by TiNP treatment; reduction in two sperm motion parameters (motile percent and progressive percent) and sperm numbers in cauda epididymides was seen. We observed Ti accumulation in the liver but not in the testis, as well as no change in plasma levels of sex hormones related to spermatogenesis. Our findings indicate that the testis is highly sensitive to TiNPs, as compared to the liver. We believe that, when considering the biological effects of TiNPs, testicular function (especially motility ability) may be a sensitive indicator.

We aimed to evaluate the effects of the length of single-walled carbon nanotubes (SWCNTs) on pulmonary toxicity in rats. Each rat received a single intratracheal instillation of short (S-) (average length of 0.40 μm) or long (L-) (average length of 2.77 μm) SWCNTs at a dose of 1 mg/kg and was observed for the next 6 months. Neither S- nor L-SWCNTs affected clinical signs, body weight, or autopsy findings. An increase in lung weight was observed after instillation of S- or L-SWCNTs; however, lung weights were slightly higher in the rats that were administered the S-SWCNTs. Distinct differences in bronchoalveolar lavage fluid (BALF) composition were observed between the S- and L-SWCNT-treated rats as early as 7 days after the intratracheal instillations of the SWCNTs. The S-SWCNTs caused persistent lung injury and inflammation during the 6-month observational period. However, the L-SWCNTs induced minimal lung injury and inflammation. Although the S- and L-SWCNTs changed BALF parameters and histopathological features of the lung, the magnitudes of the changes observed after the S-SWCNT treatment were greater than the respective changes observed after the L-SWCNT treatment. These findings indicate that the severity of the pulmonary toxicity caused after intratracheal instillation of SWCNT depends on the length of the SWCNTs. It appears that shorter SWCNTs induce greater pulmonary toxicity than longer SWCNTs do.

Although gadolinium (Gd)-based contrast media have been found to be nephrotoxic, their nephrotoxicity, and the dependence of nephrotoxicity on chelate types, have not been assessed in patients with normal or mildly diminished renal failure. This prospective, randomized study compared the nephrotoxicity of low doses of the nonionic Gd-based contrast medium gadodiamide (Omniscan®) and the ionic Gd-based contrast medium gadopentetate (Magnevist®) in patients with serum creatinine < 1.6 mg/dL. Patients aged 20 to 80 years, weighing 45 to 70 kg and with normal or < 1.6 mg/dL Serum-creatinine in the 3 months prior to undergoing magnetic resonance imaging (MRI) of brain, were enrolled. Patients were randomized to receive 0.1 mol/kg gadodiamide or gadopentetate. Serum-creatinine, serum cystatin-C, estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease (MDRD) formula, and estimated creatinine clearance rate (eCCr) using the Cockcroft-Gault formula were measured just before and 16-80 hr after MRI. Groups were compared statistically by Mann-Whitney U-tests and Wilcoxon signed-rank tests. There were no significant differences in clinical characteristics between the gadodiamide (n = 43) and gadopentetate (n = 59) groups. Serum-creatinine, eGFR and eCCr before and 16-80 hr after MRI did not differ significantly within either group or between the two groups. Serum cystatin-C was significantly higher 16-80 hr after than before MRI only in the gadodiamide group (0.79 ± 0.21 vs. 0.74 ± 0.14 mg/L, p = 0.028). The ionic contrast medium, gadopentetate, did not affect renal function during MRI, whereas the nonionic contrast medium, gadodiamide, affected renal function transiently.