Fundamental Toxicological Sciences

2017 - Vol. 4 No. 5

2017 - Vol. 4

In vitro genotoxicity test package of antibiotics for human use submitted to the Japanese regulatory agency during 2004–2015 Vol.4, No.5, p.241-245
Shin-ichi Sekizawa , Yukiko Hoshino , Aiko Takasu
Released: October 18, 2017
Abstract Full Text PDF[306K]

The Ames test is used for the mutagenic assessment of drugs; however, it may not provide an accurate genotoxic profile for bactericidal compounds. This study was performed to clarify 1) whether the total number of genotoxicity assays performed (#Assays) was greater during antibiotic development than during the development of other drugs, particularly antivirals, possibly due to the requirement for additional assessments, 2) whether the maximum doses of the Ames test were less when an alternative assay had been performed for antibiotics, and 3) whether some particular alternative assay had an advantage to minimize #Assays in the last decade. Genotoxicity data submitted to the Pharmaceuticals and Medical Devices Agency in Japan during 2004 –2015 were used. The #Assays was greater and the maximum doses of the Ames tests were lower for antibiotics, which was more obvious when alternative mutagenic assays had been performed. The mouse lymphoma assay or hypoxanthine-guanine phosphoribosyl transferase gene mutation assay was performed preferentially as an alternative. For antibiotic development, preferred genotoxicity test packages should be discussed in the future for a better understanding of the genotoxic potential of antibiotics.

Original Article
CYP3A4 induction mechanism of polycyclic aromatic hydrocarbons differs from that of rifampicin in PXR binding element Vol.4, No.5, p.229-239
Yusuke Aratsu , Reo Odagiri , Rie Shoji , Kouki Watanabe , Takeshi Kumagai , Sawako Shindo , Takamitsu Sasaki , Kiyoshi Nagata
Released: October 05, 2017
Abstract Full Text PDF[909K]

CYP3A4 is an important drug-metabolizing enzyme induced by various compounds causing drug-drug interactions. However, the molecular mechanism of CYP3A4 induction is not completely understood. CYP3A4 induction is caused by pregnane X receptor (PXR) through binding to some PXR binding elements. These elements comprise an everted repeat separated by six nucleotides in the promoter region and distal nuclear receptor binding element 1 (dNR-1) as well as the essential distal nuclear receptor binding element for CYP3A4 induction (eNR3A4) in the enhancer region of the CYP3A4 gene. Recently, we found that polycyclic aromatic hydrocarbons including anthracene induce CYP3A4 in HepG2 cells with a different induction profile from that of rifampicin (RF), a typical PXR ligand. When a CYP3A4 reporter plasmid in which the eNR3A4 DNA fragment binds directly to the CYP3A4 promoter (-362 bases) was evaluated in a reporter assay, dibenz[a,h]anthracene (DBA) induced reporter activity, while RF did not. To be induced reporter activity by RF, more 14 nucleotides 5′ upstream of the eNR3A4 (rifampicin eNR3A4: reNR3A4) DNA fragment were required. However, eNR3A4 and reNR3A4 did not respond to recombinant PXR without dNR-1. These results suggest that eNR3A4 and reNR3A4 are necessary for CYP3A4 induction by DBA and RF, respectively, and that dNR-1 is indispensable for full induction through PXR.

Original Article
A 4-week oral gavage toxicity study of L-citrulline in rats Vol.4, No.5, p.219-227
Masahiko Morita , Tsuyoshi Yamaguchi , Koji Morishita , Ayako Kamimura
Released: September 27, 2017
Abstract Full Text PDF[680K]

The safety of L-citrulline was investigated in male and female Sprague-Dawley rats by oral gavage administration for 4 weeks at a dose level of 2,000 mg/kg/day. Animals were sacrificed following the administration period. In the results, there were no toxicologically significant changes in general condition, clinical observations, body weight, food consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, or necropsy. In histopathological evaluation, squamous cell hyperplasia in the limiting ridge in the stomach was observed in some males and females in the test group. However, the lesion was limited and that tissue is specific to rodents. Thus it was considered to be less toxicologically significant. Our results indicate that repeated oral administration with L-citrulline under the present experimental conditions is well tolerated in male and female rats.

Original Article
Toxicity in repeated 28-day oral administration of N-phenyl-1-naphthylamine in rats Vol.4, No.5, p.207-218
Shihori Tanabe , Masahito Ohara , Masaya Ito , Atsushi Noda , Katsumi Kobayashi , Mariko Matsumoto , Akihiko Hirose
Released: September 13, 2017
Abstract Full Text PDF[211K]

To assess the toxicity of N-phenyl-1-naphthylamine, Sprague Dawley rats were repeatedly administered with the chemical by oral gavage daily at doses of 0, 4, 20, 100, and 500 mg/kg/day for 28 days, followed by a 14-day recovery period. A significant decrease or decreasing trend of red blood cell counts, hemoglobin concentration, hematocrit, and mean corpuscular hemoglobin concentration and a significant increase in reticulocyte counts were observed at a dose of 500 mg/kg in both male and female rats. Increase in blood urea nitrogen and sodium levels was observed in male rats that received 500 mg/kg; increase in serum total protein, albumin, and calcium levels and in albumin/globulin ratio were observed in female rats that received 500 mg/kg. Increase in relative liver weight in female rats that received 100 mg/kg and increase in the absolute and relative liver weights in both male and female rats that received 500 mg/kg were observed; increases in the absolute and relative spleen weights and absolute kidney weight in female rats that received 500 mg/kg were observed. Hypertrophy of centrilobular hepatocyte and extramedullary hematopoiesis in the spleen were observed in both male and female rats at doses of 100 and 500 mg/kg. Renal tubular dilatation and papillary necrosis were observed in both male and female rats that received 500 mg/kg. These changes had the reversible trend in the recovery period. Based on these results, the no-observed-effect-level of N-phenyl-1-naphthylamine after a repeated daily oral administration for 28 days was determined to be 20 mg/kg/day for both sexes.

Organophosphorus insecticide dichlorvos inhibits fatty acid amide hydrolase in the male reproductive organs of rats Vol.4, No.5, p.201-205
Naoko Oya , Yuki Ito , Michihiro Kamijima
Released: August 30, 2017
Abstract Full Text PDF[1M]

Organophosphorus (OP) insecticides are used worldwide to protect agricultural crops and dwellings. These chemicals phosphorylate diverse serine hydrolases, including acetylcholinesterase. Among them, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), components of the endocannabinoid signaling system (ECS) in male reproductive organs, are candidate targets of OP insecticide-induced spermatotoxicity. The effects of OP insecticides on the ECS in male reproductive accessory organs have not yet been investigated. In the present study, we examined the potential inhibitory effect of dichlorvos (DDVP) against FAAH and MAGL in male reproductive organs. In vitro screening assays were conducted by activity-based protein profiling with a fluorophosphonate chemical probe using samples of the testis, epididymis, prostate, and seminal vesicle of Wistar rats. Ex vivo assays were then performed using organs from rats orally administered 0, 5, or 10 mg/kg DDVP, 6 days per week for 9 weeks. In vitro assays showed that DDVP inhibited FAAH in the proteomes of rat testis, epididymis, and prostate, but scarcely inhibited MAGL. DDVP failed to inhibit FAAH and MAGL in the seminal vesicles. Ex vivo assays confirmed inhibition of FAAH in the proteomes of the prostate, testis, and epididymis of DDVP-treated rats, which exhibited morphologically abnormal sperm and decreased sperm motility. In conclusion, DDVP reduced the activity of FAAH but not of MAGL in male reproductive organs excluding the seminal vesicle; prostate involvement was demonstrated for the first time. Endocannabinoid signaling inhibition in these organs might contribute to sperm abnormality via deterioration in seminal plasma quality.

Toxicomics Report
Expression levels of 39 Cyp mRNAs in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a – strong expression of Cyp1b1 Vol.4, No.5, p.195-200
Satoshi Yamaori , Rongrong Jiang , Chikako Maeda , Risa Ogawa , Hiroyuki Okazaki , Hironori Aramaki , Kazuhito Watanabe
Released: August 24, 2017
Abstract Full Text PDF[184K]

An analysis of mRNA levels of 39 Cyp enzymes in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a was performed using a real-time reverse transcriptase-polymerase chain reaction. Relative expression levels were quantified by normalized β-actin levels, and compared to the mRNA expression level of the cannabinoid receptor (CB1R), which is abundantly expressed in the mouse brain. Mean 2^-ddCts of CB1R in mouse brain, C-1300N18, and NB2a cells were 1.043, 1.003, and 1.005, respectively. Among Cyp mRNAs in the mouse brain, Cyp1b1 mRNA was the most abundantly expressed (2^-ddCt = 0.310), followed by Cyp46a1 mRNA (0.246). The other Cyp mRNAs moderately expressed (0.011 ~ 0.117) were Cyp1a1, 1a2, 2b10, 2c29, 2c50, 2d9, 2d10, 2d12, 2d22, 2d26, 3a11, 3a41, 4f14, 4f15, 4f16, and 4x1. On the other hand, 10 out of 39 Cyp mRNAs (Cyp 2b9, 2b13, 2b19, 2c37, 2c38, 2c55, 3a44, 4a12, 4a14, and 4f18) were not detectable (2^-ddCt < 0.001). In the neuroblastoma cell lines, C-1300N18 and NB2a, Cyp1b1 mRNA was also the most abundant and preferentially expressed, and relative expression levels to CB1R were 4.674 and 5.084, respectively. Thirteen other Cyp mRNAs (Cyp1a1, 1a2, 2a5, 2b10, 2c44, 2c50, 2c55, 2d10, 2d22, 3a11, 4f13, 4f15, and 4f16) were detected in the neuroblastoma cell lines, whereas 17 Cyp mRNAs (Cyp2c29, 2c37, 2c39, 2c40, 2d12, 2d34, 2e1, 3a16, 3a25, 3a41, 3a44, 4a10, 4a12, 4a14, 4f14, 4x1, and 46a1) were not under the current conditions. The pattern of Cyp mRNA expression was similar for both neuroblastoma cell lines. The present results provide fundamental and useful information on the significance of particular Cyp enzymes in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a, which may be valuable tools for investigations on the neural expression and function of Cyp1b1.