Fundamental Toxicological Sciences

2021 - Vol. 8

2021 - Vol. 8

Original Article
Safety evaluation of 2-aza-8-oxohypoxanthine by in vitro skin sensitization and human tests Vol.8, No.4, p.123-133
Hisae Aoshima , Takunori Matsumoto , Rinta Ibuki , Hirokazu Kawagishi
Released: September 10, 2021
Abstract Full Text PDF[1M]

2-Azahypoxanthine (AHX) is a compound isolated from the agaricomycete Lepista sordida that forms fairy rings and 2-aza-8-oxohypoxanthine (AOH) is a metabolite of AHX in plants. In the present study, we assessed the safety of AOH for cosmetic applications via in vitro skin sensitization tests and human skin sensitization, phototoxicity, and photosensitization assays. In the tests, AOH did not induce skin sensitization in both human cell line activation test (h-CLAT) and KeratinoSensTM methods. AOH also did not induce human dermal phototoxicity or photosensitivity. Moreover, in repeat insult patch tests, the compound did not induce any human skin reaction. Hence, we conclude that AOH is safe as a cosmetic ingredient. To the best of our knowledge, this investigation is the first to evaluate the safety of AOH on human skin.

Original Article
Health risk assessment on mercury, cadmium and lead in marketed cigarettes Vol.8, No.4, p.117-122
Quang Phan Dinh , Sylvester Addai-Arhin , Randy Novirsa , Huiho Jeong1 , Willy Cahya Nugraha , Pham Hung Viet , Nobuaki Tominaga , Yasuhiro Ishibashi , Koji Arizono
Released: September 08, 2021
Abstract Full Text PDF[758K]

The Hg, Cd, and Pb concentrations in marketed cigarettes from South Korea, Vietnam, Japan, Indonesia, Taiwan, Thailand, United Kingdom (UK), Belgium, Italy, Finland, and France were investigated. The average Hg concentration in cigarettes marketed in Vietnam and Thailand had the highest trend. Meanwhile, there was more Cd found in cigarettes from Thailand, UK, and Belgium. The Pb concentrations in cigarettes from Belgium, UK, and Korea were higher than in others. In the health risk assessment in this study, the significant non-carcinogenic health risk (HI) values of Hg, Cd, and Pb were investigated. The results showed that the HI of Hg, Cd, and Pb were 4.12 × 10−2, 4.07 × 101, and 9.78 × 100, respectively. It indicated that only Cd and Pb had a significant HI. When the incremental lifetime cancer risk (ILCR) was estimated, the ILCRs for both Cd (7.32 × 10−4) and Pb (0.88 × 10−5) in cigarettes were higher than the acceptable limit. The acceptable and significant cancer risks for Pb and Cd, respectively were evaluated in cigarettes used in this study.

Original Article
Repeated-dose and reproductive/developmental toxicity screening of polyoxymethylene in rats Vol.8, No.4, p.103-116
Mariko Matsumoto , Sakiko Fujii , Nozomu Hirose , Takako Iso , Yoshiyuki Shigeta , Yasumasa Murata , Kaoru Inoue , Akihiko Hirose
Released: August 27, 2021
Abstract Full Text PDF[902K]

>The Japanese government requires risk assessment of chemicals under the Chemical Substances Control Law (CSCL). Toxicity data for polyoxymethylene (paraformaldehyde; CAS No.: 30525-89-4) for human health are insufficient though the chemical needs a screening assessment under the CSCL. Thus, polyoxymethylene was selected by the Safety Examination of Existing Chemicals and Safety Programmes of the Ministry of Health, Labour and Welfare (MHLW) to assess repeated-dose and reproductive/developmental toxicity. A combined toxicity screening was conducted following the OECD TG422. Male and female rats were administered the test chemical once daily by gavage at doses of 0 (control), 20, 60, or 200 mg/kg bw from 14 days before mating for a total of 28 to 61 days. The 200 mg/kg bw/day dose caused a significant decrease in food consumption. Histopathological examination found ulcers in the forestomach and glandular stomach, and erosion and inflammatory cell infiltration in the submucosa of the glandular stomach at the end of dosing in both sexes. Inflammatory cell infiltration in the submucosa of the glandular stomach was also observed in both sexes after the recovery period. No reproductive and developmental toxicity was observed even at the highest dose. A no-observed-adverse-effect level (NOAEL) for repeated-dose toxicity was 60 mg/kg bw/day, and a NOAEL for reproductive and developmental toxicity was 200 mg/kg bw/day, the highest dose tested.

Letter
Antidiabetic agent did not impair spermatogenesis in spontaneously hyperglycemic and diabetic rats Vol.8, No.4, p.97-101
Taiki Kobayashi , Takasumi Shimomoto , Junichi Namekawa , Masanobu Kanou , Hirotsugu Kato , Seishiro Sakamoto , Takeshi Iijima , Hideharu Ochiai
Released: August 02, 2021
Abstract Full Text PDF[849K]

In previous studies, we suggested that hypoglycemia induced by antidiabetic drugs causes testicular toxicity. In this study, we evaluated whether spontaneously hyperglycemic and diabetic rats (Goto-Kakizaki rats) treated with an antidiabetic drug showed testicular histopathological changes. TMG-123, a glucokinase activator, was given to the rats for 4 weeks at 12.5, 25 and 50 mg/kg. The exposures of TMG-123 at 50 mg/kg were similar to those that caused hypoglycemia and testicular toxicity in SD rats, and the decrease in blood glucose levels on day 28 of dosing was similar to that in SD rats, which showed that the pharmacological action of TMG-123 was similar in both SD and Goto-Kakizaki rats. However, blood glucose levels in Goto-Kakizaki rats were originally much higher than those in SD rats, and hypoglycemia was not induced in the Goto-Kakizaki rats. The histopathological evaluation showed no testicular changes. These results corroborate our hypothesis.

Original Article
Inorganic polyphosphate modulates leukocyte accumulation and vascular endothelial cell permeability and ameliorates cecal ligation and puncture-induced lethality Vol.8, No.3, p.89-96
Yoshitaka Yamazaki , Mikako Terashima-Hasegawa , Atsufumi Manabe , Toshikazu Shiba , Yumi Kawazoe , Takashi Ashino , Masahiro Hosonuma , Satoshi Numazawa
Released: July 27, 2021
Abstract Full Text PDF[1004K]

Inorganic polyphosphates with an average degree of polymerization of 150 (polyP150) have been shown to improve mortality in a lipopolysaccharide model of sepsis in mice. We aimed to investigate the effects of polyP150 in a mouse model of cecal ligation and puncture (CLP) peritonitis, which accurately reflects clinical sepsis, and elucidate its mechanism of action and suitability as a candidate for sepsis treatment. The present study demonstrated that treatment with polyP150 significantly improved survival rate in mouse model of CLP peritonitis. polyP150 inhibited a CLP-mediated increase in pulmonary vascular permeability as demonstrated by Evans blue dye assay. Pretreatment of polyP150 in human vascular endothelial cells, HMEC-1 cells, showed inhibition of tumor necrosis factor-α-induced monocytic THP-1 cell adhesion and intercellular adhesion molecule 1/CD54 gene expression. These results suggest that polyP150 ameliorates fatal sepsis by inhibiting expression of the cell adhesion molecule and the accumulation of leukocytes in the vascular endothelium, thereby suppressing the increase in vascular permeability. Our results in this study suggest that polyP150 could be a candidate for novel sepsis treatments.

Data Report
Movento® 240SC (Spirotetramat) and Envidor® 240SC (Spirodiclofen) keto-enol insecticides induce DNA damage in Drosophila melanogaster ovaries Vol.8, No.3, p.81-88
Berenyce González-Marín , María Elena Calderón-Segura , Ana Karen González Pérez , Luis Gerardo Moreno Ciénega
Released: July 27, 2021
Abstract Full Text PDF[923K]

Movento® 240SC and Envidor® 240SC are new insecticide derivatives of tetramic acid belonging to a keto-enol pesticide family. However, few studies have reported genotoxic effects in nontarget organisms. In the present study, the genotoxic effects of Movento® 240SC and Envidor® 240SC on Drosophila melanogaster ovaries were analyzed using the alkaline comet assay. Simultaneously, we determined the LD50 for both insecticides. Virgin females were exposed to food at three sublethal concentrations (11.2, 22.4, 37.3 mg/L) of Movento® 240SC and (12.3, 24.6, 41.1 mg/L) of Envidor® 240SC for 72 hr. As a negative control group, females were exposed to food without insecticides, and as a positive control group, females were exposed to 17.5 mg/L bleomycin under the same experimental conditions. We analyzed three genotoxic parameters, tail length, tail moment, and tail intensity, in ovarian cells. The results showed that 11.2 mg/L Movento® 240SC insecticide significantly increased the tail intensity mean in ovarian cells compared with the negative control. However, 22.4 and 37.3 mg/L Movento® 240SC significantly increased tail length and tail moment means compared with the negative control. Envidor® 240SC insecticide at 12.3, 24.6, 41.1 mg/L significantly increased the three genotoxic parameters in ovarian cells compared with the negative control. The LD50 values of Movento® 240SC and Envidor® 240SC insecticides were 79.1 mg/L and 78.0 mg/L, respectively. The genotoxic response of the two keto-enol pesticides was dependent on the concentration of each pesticide. The results demonstrated that Movento® 240SC and Envidor® 240SC keto-enol insecticides are genotoxic agents in D. melanogaster ovaries.

Original Article
Effects of the ethanol extract of Neopyropia yezoensis, cultivated in the Seto Inland Sea (Setonaikai), on the viability of 10 human cancer cells including endocrine therapy-resistant breast cancer cells Vol.8, No.3, p.75-80
Shuso Takeda , Masayo Hirao-Suzuki , Yukimasa Yamagishi , Takahiro Sugihara , Masataka Kaneko , Genki Sakai , Tetsuya Nakamura , Yuhzo Hieda , Masufumi Takiguchi , Masahiro Okada , Narumi Sugihara
Released: June 23, 2021
Abstract Full Text PDF[1M]

Here, we established an ethanol extraction method and obtained extracts of Neopyropia yezoensis cultivated in three different locations (extracts A-C) in the Seto Inland Sea (Setonaikai). The effects of the extracts on 10 human cancer cells derived from seven different organs were investigated. Extract A exerted the strongest anti-proliferative effects on all types of cancer cells, including an endocrine therapy-resistant aggressive breast cancer model, LTED cells. We analyzed the effects of the extracts on MCF-7 (parental cells for producing LTED cells)/LTED cells, along with four established anti-proliferative agents (etoposide, LY2835219, paclitaxel, and trichostatin A) with different action mechanisms. The inhibitory effects of extract A on both breast cancer cells were comparable with those of paclitaxel, although the other agents showed a preferable reduction in MCF-7 cell viability. We provide evidence of the involvement of component(s), especially those of extract A of N. yezoensis, which exerted anti-proliferative effects on cancer cells.

Letter
Evaluation of the in vitro cytotoxicity of oscillatoxins E and F under nutrient-starvation culture conditions Vol.8, No.3, p.69-73
Yusuke Hanaki , Yusuke Araki , Toshio Nishikawa , Ryo C. Yanagita
Released: June 15, 2021
Abstract Full Text PDF[1M]

Oscillatoxins E (1) and F (2) are cyanotoxins isolated from cyanobacteria in the genus Lyngbya. We recently reported the first total synthesis of these compounds and determined their cytotoxicity in various cancer cell lines. Their anti-proliferative activities were moderate, but 2 exhibited unique cell line selectivity. In order to understand their mode of action, in this study we evaluated the cytotoxicity of 1 and 2 under nutrient-depletion culture conditions. Interestingly, 2 exhibited stronger cytotoxicity in HHUA endometrial cancer cells, especially under FBS-starvation conditions. However, its toxicity was not increased in HHUA cells precultured in FBS-depleted medium. These results suggest that 2 is not selectively toxic to nutrient-starved cells and that FBS components such as albumin more strongly neutralized the cytotoxicity of 2 relative to 1. The protein composition of FBS varies by production lot, and the amount of FBS supplemented to culture medium is flexibly determined depending upon the cell line used and experimental objectives. Therefore, it is important to consider the detoxification activity of FBS to precisely evaluate the properties of oscillatoxins, including cytotoxic potency, cell line selectivity, and their respective structure–activity relationships.

Original Article
Effects of hemoglobin on post-mortem oxidation of bromazepam Vol.8, No.2, p.61-67
Yoshikazu Yamagishi , Hirotaro Iwase , Yasumitsu Ogra
Released: May 27, 2021
Abstract Full Text PDF[1M]

Benzodiazepines are widely used psychoactive drugs, and have been detected in several clinical cases of accidental exposure and suicide. It was reported that benzodiazepine concentration was changed in post-mortem blood. However, there is no concrete evidence to reasonably explain why benzodiazepine concentration in post-mortem blood cannot be accurately determined. In this study, we showed that the concentrations of almost all types of benzodiazepines examined were significantly decreased in the presence of hemoglobin (Hb) in vitro. In particular, bromazepam was hardly recovered in its intact form. We detected bromazepam metabolites with Hb by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra showed that bromazepam was metabolized into 3-hydroxybromazepam. Our results suggest that 3-hydroxybromazepam was formed via the Fenton reaction with the divalent iron ion in Hb. Furthermore, 3-hydroxybromazepam was also detected in post-mortem blood of autopsied subjects who intentionally ingested bromazepam, and its concentration increased with time after death. These results suggest that 3-hydroxybromazepam is a potential biomarker of bromazepam poisoning to estimate the amount of bromazepam ingested.

Original Article
3’, 4’-Dihydroxyflavone enhances all-trans retinoic acid-induced superoxide-generating activity through up-regulating transcription of gp91-phox in human monoblastic U937 cells, as opposed to flavone and other hydroxyflavone derivatives Vol.8, No.2, p.53-59
Hidehiko Kikuchi , Kaori Harata , Sumiko Akiyoshi , Harishkumar Madhyastha , Futoshi Kuribayashi
Released: May 27, 2021
Abstract Full Text PDF[1M]

Flavones are belonging to flavonoids group and show diverse biological functions. Therefore, they have much attention as drugs for maintaining human health via contributing prevention and treatment of various diseases like cancers, diabetes, neurodegenerative diseases, ischemic stroke, inflammation diseases and cardiovascular diseases. On the other hand, human monoblastic leukemia U937 cells have been used as an excellent in vitro model system for macrophage development induced in response to various reagents such as all-trans retinoic acid (RA). Here, we investigated the effects of flavones (flavone and its hydroxy derivatives) on the RA-induced O2--generating activity of U937 cells. Very interestingly, at a concentration of 20 μM, 3’, 4’-dihydroxyflavone caused up-regulation of the RA-induced O2--generating activity (to ~ 170%) although flavone and other hydroxyflavone derivatives tested showed remarkable inhibitory effects on the RA-induced O2--generating activity. The promoting effects of 3’, 4’-dihydroxyflavone on the RA-induced O2--generating activity showed the maximum value at a concentration of 10 μM. Semiquantitative RT-PCR and immunoblotting revealed that 10 μM 3’, 4’-dihydroxyflavone up-regulates the RA-induced O2--generating activity via enhancing gene expression of gp91-phox (mRNA level: to ~ 160%, protein level: to ~ 200%) while 10 μM 5, 7-dihydroxyflavone and 10 μM 3’, 4’, 5, 7-tetrahydroxyflavone down-regulate the RA-induced O2--generating activity via inhibiting gene expression of gp91-phox and p47-phox. These findings also showed that there may be various risks involved in use of phytochemical mixtures.

Data Report
Acrylamide in dog food Vol.8, No.2, p.49-52
Kazutoshi Sugita , Junpei Yamamoto , Kimika Kaneshima , Chika Kitaoka-Saito , Masashi Sekimoto , Osamu Endo , Yukihiko Takagi , Yuko Kato-Yoshinaga
Released: May 21, 2021
Abstract Full Text PDF[750K]

This study reported acrylamide, a carcinogenic substance produced by the Maillard reaction, in dog food, as a part of understand the mechanism of canine carcinogenesis. These results indicated that the average concentrations of acrylamide in dry, retort, and canned dog food were 39.6, 11.0, and 10.7 ng/g, respectively. Among the three, dry dog food exhibited significantly higher concentration. The daily intake of acrylamide by dogs was calculated to be 590 ng/kg/day, which is approximately four times higher than that of humans.

Original Article
A 90-day oral repeated-dose toxicity study of Monascus Color Y-001 in rats Vol.8, No.2, p.37-47
Yuko Doi , Taiki Sugiyama , Akihiro Hagiwara , Norio Imai , Yukinori Mera , Toyohiko Aoki
Released: May 21, 2021
Abstract Full Text PDF[785K]

Monascus Color Y-001, a natural food dye produced from Monascus purpureus fermentation, was administered orally by gavage to male and female SD rats for 90 days at doses of 0 (vehicle: 0.1% Tween 80, 10 mL/kg bw), 100, 300 and 1000 mg/kg/day. During the treatment period, there was no death, and test article effects on clinical signs were limited to reddish feces, soiled perineal region (reddish color) and salivation that were observed in both sexes at 300/1000 mg/kg/day. Prolongation in PT and APTT occurred in males at 1000 mg/kg/day, and the changes were without any evidence suggesting hemorrhage and/or hepatic dysfunction. Treatment-related histopathological findings were noted in thymus, liver and kidney, and were limited to the females at 1000 mg/kg/day. These findings included decreased cellularity in thymus with decreased thymus weights attributed to nonspecific stress, centrilobular hepatocellular hypertrophy with increase of liver weights attributed to adaptive change, and vacuolation of proximal tubules in kidneys accompanied with related parameter changes in urinalysis. From these results, the no-observed-adverse-effect level (NOAEL) was judged to be 300 mg/kg/day both in male and female rats.

Data Report
A survey on the cadmium contamination in brown rice sold in Tokyo Vol.8, No.2, p.33-36
Yukino Segawa , Setsuko Tabata , Izumi Hirayama , Kenji Iida , Ikuko Matsuno , Hisako Nakano , Takeo Sasamoto , Toshiyuki Kaji
Released: April 22, 2021
Abstract Full Text PDF[876K]

Heavy metals are ubiquitous in the environment and nature, and even in trace amounts, chronic exposure to them can have negative health effects on humans. It is known that rice, in particular, easily accumulate cadmium (Cd). Cd can accumulate in the human body and affect human health. In Japan, rice is a staple food and a main leading source of Cd poisoning. The Tokyo Metropolitan Government has been investigating the Cd content in brown rice sold in Tokyo since 1973 in order to prevent Cd poisoning in humans. A survey result from 2010 to 2018 stated that there was no sample that exceeded the maximum limit (0.4 ppm). Moreover, compared with past survey reports in Tokyo, the Cd content in brown rice has obviously decreased. In this survey, cadmium intake from brown rice was not particularly problematic in terms of food hygiene.

Original Article
The influence of long-term ingestion of D-allulose in hypercholesterolemia patients under statin therapy Vol.8, No.1, p.23-31
Misuzu Tanaka , Akane Kanasaki , Noriko Hayashi , Tetsuo Iida , Koji Murao
Released: April 22, 2021
Abstract Full Text PDF[833K]

D-allulose is a non-caloric natural sugar with health benefits. A few clinical trials with continuous D-allulose intake have been reported; one indicated significant increase in low-density lipoprotein cholesterol (LDL-C) levels, though the study was not a randomized controlled trial. D-allulose is predicted to be widely used in the near future by various people; therefore, the influence of D-allulose on those who have high risk for LDL-C elevation needs to be determined. Here, the effects of D-allulose on LDL-C levels in patients with hypercholesterolemia under statin therapy were investigated in a randomized controlled trial. Twenty subjects were randomly assigned to two groups: 15 g D-allulose/day or 15 g erythritol/day (placebo); each subject consumed a daily test substance for 48 weeks. Clinical examinations were performed every eight weeks, from initial consumption until week 52. No significant increase in LDL-C was observed, although significant decrease was observed in high-density lipoprotein cholesterol (HDL-C) in the D-allulose group. HDL-C values stayed within the standard ranges during the consumption period, and the mechanism was reported to be anti-atherosclerotic. In terms of risk assessment, D-allulose did not affect all risk factors that were measured for atherosclerotic cardiovascular disease. Taken together, these results suggested that long-term D-allulose consumption did not affect LDL-C values and atherosclerotic cardiovascular disease risk in patients with hypercholesterolemia under statin therapy.

Letter
Ouabagenin, an aglycone of cardiotonic steroid ouabain, functions as LXR ligand but avoids the increase in the SREBP-1 by inducing Krüppel-like factor 15 Vol.8, No.1, p.17-22
Tomofumi Fujino , Kouta Sugizaki , Saki Ohkawa , Sana Fujikawa , Toshiyuki Oshima , Makio Hayakawa
Released: March 27, 2021
Abstract Full Text PDF[1M]

Liver X receptor (LXR)-alpha and LXR-beta are nuclear receptors activated by oxysterols. They exhibit differential expression patterns and may perform different functional roles. Here we show that LXR-alpha and LXR-beta mutually regulate the expression levels of their counter parts in the normal hepatocyte-derived cell line Fa2N-4. In addition, we demonstrate that ouabagenin (OBG), which was identified as a naturally occurring LXR ligand without causing hepatic steatosis, dramatically increases the expression of LXR-alpha in Fa2N-4 cells that overexpress LXR-beta. However, the expression level of sterol response element binding protein 1c (SREBP-1c), a known target of LXR-alpha, remains marginal in OBG-treated Fa2N-4 cells, in which LXR-alpha expression is upregulated by LXR-beta. Furthermore, we show that OBG stimulates the expression of Krüppel-like factor 15 (KLF15) that is known to form a repressive complex with LXR/RXR and corepressor RIP140, thereby reducing SREBP-1c expression. Thus, we propose a novel mechanism that OBG avoids the increase in the expression of SREBP-1c through the upregulation of KLF15.

Original Article
Genotoxicity of Monascus Color Y-001 Vol.8, No.1, p.7-16
Ryosuke Sato , Michihito Takabe , Takahiro Ishii , Norio Imai , Yuko Doi , Toyohiko Aoki
Released: March 09, 2021
Abstract Full Text PDF[747K]

The genotoxic potential of Monascus Color Y-001 was assessed using the standard battery of assays including the in vitro reverse mutation test in bacteria (Ames test), the in vitro chromosomal aberration test in mammalian cells and the in vivo micronucleus test in rats. The results of the Ames test, the chromosomal aberration test in CHL/IU cells with and without S9 mix (metabolic activation) and the in vivo bone marrow micronucleus test in rats were all negative. Therefore, it is concluded that Monascus Color Y-001 does not possess any genotoxic risk in humans.

Toxicomics Report
Comprehensive analysis of the alteration of plasma miRNA expression level in mice exposed to diesel exhaust Vol.8, No.1, p.1-6
Ken Tachibana , Iori Kodaira , Noriko Kuroiwa , Ryo Uzuki , Yusuke Shinkai , Ken Takeda
Released: February 12, 2021
Abstract Full Text PDF[768K]

MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides in length that play important roles in controlling a huge range of eukaryotic cell functions. Many studies have shown that abnormal expression levels of miRNAs are associated with many diseases and detrimental health effects caused by exposure to environmental pollutants and particulate matter. As a number of reports suggest that profiles of miRNAs in body fluids reflect physiological and pathological status, extracellular miRNAs, especially in plasma and serum, are being focused on as candidate disease biomarkers. Although these phenomena suggest that expression levels of plasma miRNAs can also be used as biomarkers for the detection of adverse health effects caused by exposure to environmental pollutants, there are still few studies on this subject. In the present study, we used diesel exhaust (DE) and filtered-DE (F-DE), which is DE with the particulate matter removed, as a model for environmental pollutant exposure and comprehensively analyzed alteration of the expression levels of plasma miRNAs in mice using a LNA miRNA microarray. MiRNA microarray analyses showed altered expression level of 5 plasma miRNAs (miR-1983, miR-720, miR-1957, miR-335-3p, and miR-1897-5p) in the DE-exposed group and F-DE-exposed group. The results show both the possibility that exposure to various environmental chemicals including DE alters plasma miRNAs and the potential for plasma miRNAs to be used as biomarkers of exposure to these chemicals.