Fundamental Toxicological Sciences

2020 - Vol. 7

2020 - Vol. 7

Original Article
Safety evaluation of 2-aza-8-oxohypoxanthine based on in vitro and human patch tests Vol.7, No.5, p.207-214
Hisae Aoshima , Sayuri Hyodo , Rinta Ibuki , Jing Wu , Jae-Hoon Choi , Hirokazu Kawagishi
Released: June 19, 2020
Abstract Full Text PDF[1M]

2-Azahypoxanthine (AHX) and imidazole-4-carboxamide (ICA) are fairy-ring causing compounds from a mushroom-forming fungus, Lepista sordida, and 2-aza-8-oxohypoxanthine (AOH) is a metabolite of AHX in plants. However, the safety of AOH had not yet been elucidated. In this study, we focused on AOH and performed safety evaluations of the compound using in vitro and human patch tests for cosmetic applications. In the Ames test, AOH was not mutagenic to any of the test bacterial strains (> 5000 μg/plate). In vitro skin irritation and skin sensitization studies using reconstructed human epidermis and peptides that contained lysine and cysteine showed that AOH was not a skin irritant (cell viability > 50%) and did not exhibit skin sensitization. This compound also did not exhibit cytotoxicity under ultraviolet- or sham-irradiation in the alternative phototoxicity test using BALB/c 3T3 cells (mean photo effect < 0.1) and no skin reaction was observed in the patch test on human skin. Thus, we concluded that AOH is safe as a cosmetic ingredient. This is the first study in which safety evaluation tests were performed on AOH.

Original Article
Retinoic acid dramatically enhances cytotoxicity of equol against human monoblastic U937 cells, but not against human peripheral neutrophils Vol.7, No.4, p.201-206
Hidehiko Kikuchi , Kaori Harata , Chikage Kawai , Harishkumar Madhyastha , Akira Yamauchi , Futoshi Kuribayashi
Released: May 27, 2020
Abstract Full Text PDF[1M]

Since phytoestrogens (e.g. biochanin A, coumestrol, daidzein, genistein and glycitein) are structurally similar to estrogen, they mimic estrogen function. Phytoestrogens consist in certain edible plants, especially in soybeans. Among them, interestingly, equol that has the greatest estrogenic activity is generated from daidzein by gut microbes. Therefore, the relationship between phytoestrogens and gut (including intestinal cells and bacteria) is being watched with strong interest. In this paper, we revealed that all-trans retinoic acid (RA) dramatically enhances cytotoxicity of several phytoestrogens against U937 cells. While β-estradiol and phytoestrogens tested showed no effect on the viability of U937 cells in the absence of RA, 10 μM coumestrol, (±)-equol and genistein brought about remarkably reduced viability of U937 cells at 24 h (to ~15%, ~7% and ~35%, respectively) in the presence of 1 μM RA. In particular, the cytotoxicity of (±)-equol was drastically enhanced in the presence of RA. Moreover, very interestingly, (±)-equol showed no effect on the viability of human peripheral neutrophils even in the presence of RA. As is well known, human monoblastic leukemia U937 cells have been used as an in vitro model for macrophage that exists in intestine and plays significant roles to maintain intestinal homeostasis. These data suggest that equol not only can serve as an effective modifier in therapy for leukemia in combination with RA but also may affect the maintenance of intestinal homeostasis.

Original Article
Postnatal wheel running mitigates endocrine disruption of mammary gland development in mice Vol.7, No.4, p.189-199
Emily E. Schmitt , Weston W. Porter , J. Timothy Lightfoot
Released: May 27, 2020
Abstract Full Text PDF[4M]

We investigated if access to a running wheel after in utero exposure to benzyl butyl phthalate (BBP) ameliorates the toxicological effects of BBP exposure. Our purpose was to determine if post-birth exercise after prenatal BBP exposure could reverse alterations in mammary gland development. Twenty-five female pups were exposed to 500 mg/kg of BBP on days 9-15 in utero and analyzed for mammary gland development and morphology. Mice either had access to a running wheel beginning at 8 weeks of age or were in a cage with a “locked” wheel to prevent running activity. Whole mount staining showed delayed mammary gland morphology development, regardless of wheel exposure in the treated groups. Additional histology staining revealed BBP exposed mice that were not allowed exercise, had larger ducts with multiple cell layers containing proliferative cells suggesting a favorable environment for tumor growth. In addition, there was a significant increase in progesterone status in the mouse mammary gland at 20 weeks but not 10 weeks, regardless of wheel exposure. BBP exposure led to abnormal mammary gland development in female mice and access to a running wheel helped ameliorate some, but not all, of the harmful effects due to BBP exposure at either 10 weeks or 20 weeks of age. Our results are significant because they indicate that exercise can reverse most of the BBP-initiated alterations in the mouse mammary gland, and physical activity has a positive impact on most developmental parameters in the mouse mammary gland.

Letter
Repeated 28-day and 13-week dose toxicity studies of oils prepared from the internal organs of the Japanese giant scallop (Patinopecten yessoensis) in rats Vol.7, No.4, p.177-188
Koki Sugimoto , Eito Shimizu , Nozomi Hagihara , Ryota Hosomi , Kenji Fukunaga , Munehiro Yoshida , Takeya Yoshioka , Koretaro Takahashi
Released: May 27, 2020
Abstract Full Text PDF[959K]

The discarded internal organs of the Japanese giant scallop (Patinopecten yessoensis) are abundant resources rich in n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid and docosahexaenoic acid. However, they have not been utilized due to contamination with toxic substances such as cadmium (Cd) and the occurrence of diarrhetic shellfish toxins (DST). We have successfully prepared a high-quality scallop oil (SCO) from its internal organs with negligible contamination with Cd and DST. The scallop internal organs were obtained from two different scallop processing areas, Mutsu and Uchiura bays, Japan, and referred to as SCO-M and SCO-U, respectively. To evaluate the safeties of SCO-M and SCO-U as food ingredients and n-3 PUFA supplements, repeated 28-day and 13-week dose oral toxicity studies in rats were conducted. Rats were fed diets containing 1% and 5% of SCO-M and SCO-U, respectively, in the repeated 28-day dose oral toxicity study and 5% SCO-M or SCO-U in the repeated 13-week dose oral toxicity study (limit test). No adverse toxicological effects were observed when rats were fed diets containing SCO-M and/or SCO-U at up to 5% for 28 days and 13 weeks. These results suggest that SCO-M and SCO-U are safe products in terms of subacute toxicity under these experimental conditions.

Original Article
The aminoethyl group is a crucial structural moiety in metal-mediated oxidative DNA damage by catecholamines Vol.7, No.4, p.171-176
Koji Ueda , Yoshihiko Nishino , Yoshinori Okamoto , Nakao Kojima , Hideto Jinno
Released: May 19, 2020
Abstract Full Text PDF[1M]

Oxidative stress is involved in the development of many neurological diseases. The interactions between catecholamines and copper or iron generate reactive oxygen species that lead to oxidative DNA damage in vitro. Furthermore, catechol structure is essential for DNA damage. Here, we clarified the effect of aminoethyl side chains on DNA damage. Endogenous catecholamines (dopamine, noradrenaline, and adrenaline) were more effective than other catechols (catechol, 4-ethylcatechol, and 3,4-dihydroxybenzylamine) in strand break and base oxidation of calf thymus DNA. The presence of copper caused more DNA damage than iron. Furthermore, adrenaline oxidized to adrenochrome more rapidly by copper than iron. Leukoadrenochrome, an oxidation intermediate formed by the intramolecular cyclization of aminoethyl side chains, rapidly increased the formation of 8-hydroxy-2′-deoxyguanosine compared with adrenalin. These results show the effect of aminoethyl side chains in catecholamine-induced oxidative DNA damage. This mechanism may partly show the vulnerability of catecholaminergic neurons against oxidative stress.

Letter
Bacteriological evaluation of feasibility of food poisoning from long-term stored “pack-cooked” meals Vol.7, No.4, p.167-170
Toshihiro Kobayashi , Harumi Sakuda
Released: May 19, 2020
Abstract Full Text PDF[851K]

Recently, a “pack cooking” method, which uses heat-resistant plastic bags, has attracted attention for being simple and safe for emergencies such as natural disasters. One serving is filled in a pack. It uses less kitchenware and clean water, so “pack cooking” will particularly be a powerful tool for soup runs. However, its risk for food poisoning has not been given attention. It is important to consider the safety of “pack-cooked” meals, because medical shortage is as much a problem as food shortage during natural disasters. We conducted a bacteriological evaluation of curry and rice as typical “pack-cooked” meals, assuming that they were stored at room temperature for a long time. “Pack-cooked” meal samples were stored at 25°C and 4°C for 24 hr, and their homogenates were used as sample for bacteriological analysis. Obvious standard plate count bacteria were found in “pack-cooked” curry, which was stored at 25°C for 24 hr, whereas those stored at 4°C for 24 hr did not. Furthermore, there was a sample that was stored at 25°C for 24 hr in which coliform bacteria were detected. Since 25°C is equivalent to room temperature, our results demonstrate that the “pack-cooked” curry stored at room temperature for 24 hr could cause food poisoning. While “pack cooking” is a useful method, it is important to understand that prolonged storage at room temperature of “pack-cooked” meals should be avoided.

Letter
Combined exposure to environmental electrophiles enhances cytotoxicity and consumption of persulfide Vol.7, No.3, p.161-166
Masahiro Akiyama , Takamitsu Unoki , Yoshito Kumagai
Released: May 09, 2020
Abstract Full Text PDF[3M]

Environmental electrophiles readily interact with reactive sulfur species (RSS), resulting in decline of cellular RSS levels accompanied by formation of their sulfur adducts. In the present study, we examined the effects of combined environmental electrophiles on consumption of persulfide and on cytotoxicity in HepG2 cells. A convenient assay with SSP4, a fluorometric probe for detection of per/polysulfides, indicated that each environmental electrophile caused compound-dependent consumption of persulfide. Consumption of persulfide by combined exposure to methylmercury (MeHg) and cadmium (Cd) was greater than that of the single exposure. Consistent with this finding, combined exposure to environmental electrophiles (MeHg, Cd, and 1,4-naphthoquinone) exacerbated concentration-dependent cellular toxicity in HepG2 cells compare to single exposure to each compound. As humans are exposed to environmental electrophiles daily, more attention should be paid to the study of combined exposure to environmental electrophiles, which can modulate cellular levels of RSS and disrupt redox homeostasis.

Original Article
Usefulness of HepaRG cells in the mitochondrial function assay for the estimation of potential risk of idiosyncratic drug-induced liver injury Vol.7, No.3, p.153-160
Keisuke Goda , Taku Masuyama
Released: May 09, 2020
Abstract Full Text PDF[1M]

Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events and is classified into intrinsic and idiosyncratic types. Almost all of DILI caused in humans is known to be idiosyncratic type. The estimation of the potential risk for a drug candidate to induce idiosyncratic DILI is important to facilitate the development of new drugs, however, the estimation is difficult from the results of non-clinical toxicity studies using animals. We have previously reported the in vitro combination assay of mitochondrial function and apoptosis using human primary hepatocytes as a useful model for estimation of the risk of idiosyncratic DILI. In this study, to improve the in vitro assay for estimation of the risk of idiosyncratic DILI, we evaluate the usefulness of HepaRG cells for the mitochondrial function assay. We measured the oxygen consumption rate (OCR) as an endpoint of mitochondrial function in HepaRG cells treated with some compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac) and others known not to cause idiosyncratic DILI (acetaminophen and ethanol) and compared the results in HepaRG cells and with those for human primary hepatocytes as previously reported. As the results, HepaRG cells showed comparable or even higher sensitivity for detecting mitochondrial dysfunction than human primary hepatocytes, in all tested compounds. Taking into account these results and many other useful properties of the cells, HepaRG cells are considered to be much more suitable for this mitochondrial function assay than the human primary hepatocytes.

Original Article
Maltosyltrehalose Syrup: Bacterial reverse mutation test, and 90-day feeding and 90-day repeated oral dose toxicity studies in rats Vol.7, No.3, p.141-152
Shuji Matsumoto , Takaharu Hashimoto , Chie Ushio , Keisuke Namekawa , Alan B. Richards
Released: April 22, 2020
Abstract Full Text PDF[1M]

Maltosyltrehalose syrup (TG4 syrup) is an enzymatically derived starch hydrolysate consisting of only glucose. The main components are maltotetraose (G4) and maltosyltrehalose (TG4). G4 is a common component of essentially all starch hydrolysates, and consists of only four glucose molecules with α-1,4 linkages. TG4 also consists of four glucose units, but the terminal glycosidic bond at the reducing end of the glucose chain is inverted, which results in the final bond being α-1,1. The two terminal glucose units form a trehalose disaccharide molecule, attached to a maltose molecule. The presence of the trehalose moiety in the TG4 molecule results in unique functional and technical properties from what is found in other α-1,4 linked oligosaccharides. This paper presents results of standardized toxicity studies of TG4 syrup, including an in vitro bacterial mutagenicity test, a 90-day oral feeding study in rats, and a 90-day oral toxicity (gavage) study in rats. Treatment with TG4 syrup resulted in no microbial mutagenic activity or growth inhibition in either Salmonella typhimurium or Escherichia coli, even at the maximum dose of 5,000 μg/plate. The NOAEL in the 90-day oral feeding study was calculated as 10% of TG4 syrup in the diet, which was equal to 6,818 and 7,464 mg/kg/day (dwb) in male and female rats, respectively. The 90-day oral gavage toxicity study had a NOAEL of 5,000 mg/kg/day (dwb) in male and female rats. Taken together these data showed that TG4 syrup was safe for use in these studies, suggesting it is safe for consumption by humans.

Original Article
Sex differences in the effects of high-fat diet on mouse sciatic nerves Vol.7, No.3, p.133-139
Masahiro Ogawa , Takahiro Kimura , Yoshitaka Tanetani , Masami Hori , Takahiro Kyoya , Megumi Terada
Released: April 15, 2020
Abstract Full Text PDF[1M]

Obesity is caused by a chronic positive energy balance, which not only increases the amount of lipid in adipose tissue, but also, in non-adipose tissue. Excessive accumulation of lipids in tissues may lead to cell dysfunction or cell death, a phenomenon known as lipotoxicity. The aim of this study was to investigate the effects of high-fat diet (HFD) feeding on the sciatic nerves of both male and female mice. HFD feeding induced increased mRNA levels of Bax and Bcl2 in HFD group of males only, although the accumulation of fatty acids in the sciatic nerves was induced by HFD feeding in the both sexes. To determine whether estrogen was involved in the inhibitory effects of HFD feeding-induced increased expression of apoptosis-related genes, ovariectomized (OVX) females were fed a normal diet (ND) or HFD with or without daily ethinylestradiol treatment for 1 week. In OVX mice, the mRNA levels of Bax and Bcl2 in HFD group were higher than in ND group. In contrast, in OVX mice treated with ethinylestradiol, there was no significant between ND and HFD groups. In conclusion, HFD induced apoptosis in the sciatic nerves of males, but not females, and estrogen had inhibitory effects on HFD-induced apoptosis in the sciatic nerves of females. Long-term feeding studies are needed to investigate the pathological effects on sciatic nerves of mice fed HFD as pathological findings were not observed under our study condition.

Original Article
In vitro toxicity studies of epoxyoleic acid and diepoxylinoleic acid Vol.7, No.3, p.123-132
Takashi Kitaguchi , Masaharu Tanaka , Takahiro Matsuda , Taisei Mizota , Katsutoshi Ohno , Kazuhiro Kobayashi , Yasumitsu Ogra , Mitsuru Tanaka
Released: April 15, 2020
Abstract Full Text PDF[5M]

Epoxidized fatty acids are generated during food processing and detected in various lipid- and oil-containing foods. Although compounds with an epoxide structure have high reactivity and there is increasing concern about their potential toxic effects such as genotoxicity and cellular damage, information regarding the toxicity of epoxy fatty acids is largely unknown. Therefore, we conducted three in vitro genotoxicity studies (bacterial reverse mutation assay, in vitro micronucleus test, and p53R2-dependent luciferase reporter gene assay) and HepG2 cytotoxicity assays for epoxyoleic acid (EOA) and diepoxylinoleic acid (DELA). The in vitro genotoxicity results of EOA and DELA were uniformly negative. In the cytotoxicity assay, EOA and DELA induced weak cytotoxicity at high concentrations, but the effect was similar to those of oleic and linoleic acids at low concentrations. Considering the existing toxicity information on epoxidized soybean oil, which is a similar compound, there might be little concern concerning the health effects of epoxy fatty acids at low concentrations.

Letter
Effects of estrogen on fatty-acid-induced cytotoxicity in mouse Neuro-2a neural cells Vol.7, No.2, p.115-121
Masahiro Ogawa , Takahiro Kyoya , Takahiro Kimura , Megumi Terada
Released: March 27, 2020
Abstract Full Text PDF[1M]

The accumulation of free fatty acids induces lipotoxicity in neural cells. Estrogen, 17β-estradiol (E2) protects against the damage of cells in various organs and tissues. However, the role of E2 on lipotoxicity in neural cells remains unclear. In this study, we investigated the effects of E2 on stearic acid (saturated fatty acid)- and oleic acid (unsaturated fatty acid)-induced cytotoxicity in retinoic acid-induced mouse neuroblastoma Neuro-2a differentiated into neural cells. Cell viability was evaluated by lactate dehydrogenase release from Neuro-2a neural cells. Stearic acid and oleic acids suppressed the cell viability in a dose-dependent manner. E2 prevented oleic acid-induced cytotoxicity but had no effect on stearic acid-induced cytotoxicity. ERα-selective agonist prevented cytotoxicity in Neuro-2a neural cells. In contrast, ERβ-selective agonist slightly significantly enhanced the cytotoxicity in the presence of oleic acid. Oleic acid, but not stearic acid, increased the mRNA level of p62/Sqstm1. E2 treatment statistically significantly, but slightly, enhanced the stearic acid-induced Bax expression. In contrast, E2 and ERα-selective agonist inhibited the oleic acid-induced the p62/Sqstm1 expression. Our results suggested that fatty acids induced cytotoxicity in Neuro-2a neural cells, and estrogen prevented the oleic acid-induced cytotoxicity via ERα, but not ERβ. Further studies are needed to understand the role of ERβ in neuron injury under normal conditions.

Original Article
Evaluation of cytokine storms in a disseminated intravascular coagulation monkey model Vol.7, No.2, p.105-114
Yoshitaka Hirasawa , Atsushi Fujiwara , Kazuya Tabata , Kenji Yoshida , Tsutomu Negama , Takayuki Anzai , Shin-ichi Sato
Released: March 27, 2020
Abstract Full Text PDF[2M]

The purpose of this study was to profile cytokine storms (cytokine release syndrome) in the LPS-induced disseminated intravascular coagulation (DIC)-cynomolgus monkey model by measuring changes in 22 cytokines using Luminex. In this study, increases were noted in 20 cytokines, excluding IL-4 and IL-17A. Specifically, IL-6, IL-8, G-CSF and TNF-α, pro-inflammatory cytokines, and IL-10, an anti-inflammatory cytokine, as well as MCP-1, markedly increased by 10,000 pg/mL or more. In addition to the marked increases in the pro-inflammatory cytokines IL-6 and G-CSF, the concentrations of IL-5, IL-18, IFN-γ, VEGF and IL-15 increased continuously. Also, in addition to the marked increases in the pro-inflammatory cytokine IL-8 as well as in MCP-1, the concentrations of IL-1ra, IL-2, IL-1β, IL-12/23 (p40), GM-CSF and TGF-α gradually decreased after initially increasing. On the other hand, in addition to the marked increases in the pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10, MIP-1β and MIP-1α transiently increased and then rapidly disappeared from serum. IL-13 increased at 6 hr after administration only. Since the behavior of cytokines in this monkey model was similar to those noted in DIC in humans, this model will be useful for evaluating the efficacy of anti-DIC drugs. In addition, this model will also be useful for assessing the risk of cytokine storm development, which is a serious adverse effect of certain types of antibody drugs and CAR-T cell-based therapies.

Original Article
5-Fluorocytosine induces fetal skeletal malformations in rats by altering expression of Homeobox genes Vol.7, No.2, p.97-103
Takayuki Kumamoto , Mika Senuma , Mai Todoroki , Fumiaki Kumagai , Hajime Imai , Reiko Suzuki , Tetsuo Ogawa , Makiko Kuwagata
Released: March 27, 2020
Abstract Full Text PDF[2M]

5-Fluorocytosine (5-FC) is an antimycotic and teratogenic compound. Oral administration of 5-FC to pregnant rats on gestation days (GD) 9 and 13 was shown to induce thoracolumbar supernumerary ribs (TSR, 14th rib) and abnormal digits, respectively, in fetuses. This study investigated the effects of 5-FC on homeobox genes, which control the anterior-posterior-axis. 5-FC (75 mg/kg) was administered orally on GD9 and GD13, and tissues collected from cranial and caudal regions of TSR sites were analyzed. Following 5-FC administration on GD9, the levels of expression of Hoxa10, which determine the position of the thoracic and lumbar vertebrae, were decreased at GD13. Analysis of hindlimbs 6 hours after administration on GD13 showed decreases in expression of Hoxa11, Hoxd12, and Hoxd13, the Hox genes responsible for limb formation from the proximal to distal, and from the anterior to posterior directions. The present findings showed that altered expression of Hox genes contributes to 5-FC teratogenicity.

Original Article
Utility of measuring long bone length in toxicity studies: results of a 14-day repeated dose oral toxicity study of dexamethasone in young, periadolescent and adult rats Vol.7, No.2, p.85-96
Chihiro Noguchi , Satoshi Tsuji , Yutaka Nakanishi , Minoru Sasaki
Released: March 03, 2020
Abstract Full Text PDF[6M]

Measurement of the bone length is not used routinely in toxicity studies but used to examine growth and bone toxicity. To investigate the utility of measuring the bone length in toxicity studies and to identify the appropriate site, we evaluated femur, tibia, humerus, and sternum, in a 14-day repeated dose oral toxicity study of Dexamethasone (DEX), which is known to cause growth retardation and osteoporosis, in young, periadolescent, and adult rats. To observe the effect of decreased food consumption, we also evaluated the changes in each diet-restricted group in which the food intake restricted to levels corresponding to that consumed by the DEX-treated periadolescent and adult rats. Significant decreases of the bone length at all the measured sites and histopathological findings in growth plates and/or trabecular bone were observed in the DEX-treated young and periadolescent rats. Significant decreases of the femoral length and decreased trabecular bone were observed in the DEX-treated adult rats. No histopathological changes were observed in any of the diet-restricted groups, while decreases of the femoral length, similar to that in the DEX-treated adult rats were observed in the diet-restricted adult rats. The results suggested that measurement of the bone length in femur, tibia, humerus, and sternum was useful in young and periadolescent rats, and measurement of the femoral length was useful in adult rats. Moreover, our results showed that the decreases in the femoral length in the DEX-treated adult rats were not only related to the DEX-treatment, but were also influenced by the decreased food consumption.

Original Article
Arsenite inhibits gene expression of perlecan, syndecan-1, -2, -3 and biglycan in cultured vascular endothelial cells Vol.7, No.2, p.77-83
Dong-pan Wu , Tsuyoshi Nakano , Yayoi Tsuneoka , Tsutomu Takahashi , Yo Shinoda , Toshiyuki Kaji , Yasuyuki Fujiwara
Released: March 03, 2020
Abstract Full Text PDF[1M]

Arsenic is an environmental pollutant and is a possible risk factor for vascular diseases such as atherosclerosis. Vascular proteoglycans (PGs) are key molecules in the initiation and progression of atherosclerosis. We previously demonstrated that arsenite, but not arsenate, decreases the synthesis of both heparan sulfate proteoglycans (HSPGs) and chondroitin/dermatan sulfate proteoglycans (CS/DSPGs) in cultured vascular endothelial cells. In the present study, we aimed to identify the PG molecules whose expression is decreased by arsenite, using a culture system of bovine aortic endothelial cells. The results indicate that a 24-hr treatment of arsenite significantly decreases the mRNA levels of a large HSPG perlecan, small HSPGs—syndecan-1, -2 and -3—, and a small CS/DSPG biglycan in vascular endothelial cells without nonspecific cell damage; the expression of syndecan-4 mRNA was unaffected by arsenite. The decreased expression of perlecan, syndecan-1 and biglycan genes began after 3 hr of arsenite treatment. However, arsenate did not change the mRNA expression levels of perlecan and biglycan in the cells. These results suggest that the inhibition of synthesis by arsenite occurs in particular types of proteoglycans, i.e. perlecan, syndecan-1, -2, -3, and biglycan in vascular endothelial cells.

Original Article
Toxicological evaluation of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) Vol.7, No.2, p.55-76
Osamu Ohgoda , Ian N. Robinson
Released: March 03, 2020
Abstract Full Text PDF[1009K]

In the inhalation field, lipids such as egg phosphatidylcholine (PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and dipalmitoylphosphatidylcholine (DPPC), are considered to be generally recognized as safe (GRAS), comprising materials that are endogenous to the lungs and locally present in large quantities. Indeed, PC, DSPC and DPPC may be used to form liposomes which are known to promote an increase in drug retention time and reduce the toxicity of drugs after administration. Unfortunately, published literature guidance about the safety evaluation of these lipids as pharmaceutical excipients for use in inhaled products and about application for marketing authorization, is very limited. The purpose of this article is to review the potential toxicity of DSPC for pulmonary administration. Given the use of air and vehicle controls in a range of inhalation toxicology studies as well as negative genotoxicity and also reproductive toxicity results, it is thought that the use of DSPC is shown to be safe for pulmonary administration.

Original Article
Comparison of the liver findings after simvastatin-treatment between Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats and Sprague-Dawley rats Vol.7, No.1, p.41-54
Tadakazu Takahashi , Yusuke Suzuki , Naohito Yamada , Kaoru Toyoda , Keisuke Goda , Katsunori Ryoke , Chizuru Matsuura , Akio Kobayashi , Shoichiro Sugai , Kayoko Shimoi
Released: January 28, 2020
Abstract Full Text PDF[3M]

One of the risk factors for drug-induced liver injury (DILI) is the diabetic state. Our previous investigation showed that liver injury after repeated oral dosing with allyl alcohol and carbamazepine was enhanced more in the Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats than in the Sprague-Dawley (SD) rats. It was caused by lower hepatic detoxification due to depleted hepatic glutathione synthesis. This is because simvastatin, frequently used in diabetic patients, shows a positive high reaction in a GSH adduct assay in vitro although the GSH adduction is considered not to be a major metabolic pathway of simvastatin. Therefore, in the present study, effects of simvastatin-treatment on the liver were compared between the Sprague-Dawley (SD) rats and SDT fatty rats in order to obtain additional information to estimate the potential risk of DILI in the diabetic state. There were no effects with simvastatin on the liver in the SD or SDT fatty rats after 13-week oral dosing of simvastatin. These results indicate that simvastatin does not have potential to induce liver injury in diabetic state and are consistent with the reports for the clinical use of simvastatin in the diabetic patients.

Original Article
Triphenyltin inhibits GA-binding protein α nuclear translocation Vol.7, No.1, p.33-40
Naohiro Kidoguchi , Keishi Ishida , Seigo Sanoh , Masatsugu Miyara , Yaichiro Kotake
Released: January 23, 2020
Abstract Full Text PDF[2M]

Organotin compounds such as triphenyltin (TPT), which are common environmental pollutants, had been widely used as antifouling agents for ship bottoms. Although toxic effects of organotins through nuclear receptors such as retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) γ have been well demonstrated, other mechanisms underlying organotin-induced toxicity have hardly been reported. In the present study, we focused on the transcription factor GA-binding protein (GABP), which regulates the expression of various housekeeping genes, as a novel target of TPT toxicity. We investigated the change of GABPα subunit protein expression induced by TPT. Although 100-500 nM concentration of TPT was not found to affect the total protein expression of GABPα, TPT significantly decreased nuclear translocation of GABPα in human embryonic kidney (HEK) 293T cells. In addition, TPT increased intracellular reactive oxygen species (ROS) levels. Both inhibition of GABPα nuclear translocation and the increase in ROS levels were observed in menadione (an ROS inducer)-treated HEK293T cells. Our results indicate that TPT causes inhibition of GABPα nuclear translocation, which may be triggered by ROS production. This might have serious implications in cellular physiology, thereby affecting cell survival.

Original Article
Safety and efficacy of a 48-week long-term ingestion of D-allulose in subjects with high LDL cholesterol levels Vol.7, No.1, p.15-31
Misuzu Tanaka , Akane Kanasaki , Noriko Hayashi , Tetsuo Iida , Koji Murao
Released: January 17, 2020
Abstract Full Text PDF[894K]

D-allulose is one of the rare sugars with almost zero calories and several health benefits. Previous studies have reported the safety of D-allulose in normal, overweight/obese, and diabetic humans. However, one study reported significant increases in T-Cho and LDL-C after 12 weeks of D-allulose intake; this report was not a randomized controlled trial and these changes were considered to be due to seasonal variations. We, therefore, conducted a randomized, double-blind, placebo-controlled trial in 90 subjects with high LDL-C levels for 48 weeks to clarify the influence of long-term D-allulose consumption on cholesterol metabolism and efficacy. Subjects were randomly divided into 3 groups: high-dose D-allulose (15 g D-allulose/day), low-dose D-allulose (5 g D-allulose/day), and placebo group (0 g D-allulose/day); each subject consumed a daily test beverage for 48 weeks. Clinical examinations were performed every eight weeks, beginning from initial consumption until week 52. No significant increases in T-Cho and LDL-C between test groups were observed, and 48 weeks of D-allulose consumption did not change risk factors for atherosclerotic cardiovascular disease. Furthermore, no clinical problems were recognized for other parameters. Additionally, significant improvements in hepatic enzyme activities, fatty liver score, and glucose metabolism after long-term D-allulose consumption were observed. The results from our study revealed that 1) D-allulose consumption is considered safe for long-term intake up to a year, and 2) D-allulose may be effective for improving hepatic functions and glucose metabolism.

Letter
Usefulness and limitations of mRNA measurement in HepaRG cells for evaluation of cytochrome P450 induction Vol.7, No.1, p.9-14
Kenta Mizoi , Yuuki Fukai , Eiko Matsumoto , Satoshi Koyama , Seiichi Ishida , Hajime Kojima , Takuo Ogihara
Released: January 15, 2020
Abstract Full Text PDF[1M]

Cytochrome P450s (CYPs) are involved in the metabolism of various drugs, and may generate toxic metabolites or intermediates that result in drug-induced liver injury (DILI). Consequently, inducers of CYPs may promote DILI. In a draft test guideline, the Organisation for Economic Co-operation and Development (OECD) recommends measurement of the metabolic activity of CYP as an index for assessing CYP-inducing activity. However, change of mRNA level has also been used as a simple parameter to evaluate CYP induction. In this study, therefore, we examined the usefulness and limitations of mRNA expression measurement for evaluation of the induction of CYP1A2, CYP2B6, and CYP3A4 by omeprazole, phenobarbital, and rifampicin (RIF), respectively, in HepaRG cells, a well-established cell line derived from human hepatocellular carcinoma. The results of mRNA measurement correlated well with the results of metabolic activity measurement in the lower concentration ranges for all inducers, even though we observed significant decreases in albumin and urea secretion in the presence of 10 µM RIF, reflecting its known hepatotoxicity. Our results indicate that mRNA measurements and metabolic activity measurements in HepaRG cells generally give comparable results for fold-induction of CYPs.

Original Article
Azoxystrobin at sub-cytotoxic concentrations disrupts intracellular zinc homeostasis: A flow cytometric analysis with rat thymic lymphocytes and fluorescent probes Vol.7, No.1, p.1-7
Mai Shoji , Masaki Asada , Akihiko Matsumoto , Haruki Nishino , Ao Yi Xiang , Mizuki Mizobuchi , Naoki Kanematsu , Hajime Miura , Norio Kamemura
Released: January 10, 2020
Abstract Full Text PDF[3M]

Azoxystrobin is a broad-spectrum fungicide having a wide usage. However, the toxic effect of azoxystrobin in humans is not reported. In Japan, azoxystrobin was detected at a five-fold higher concentration than the normal upper limit (2.5 mg/kg) in a shipment of an Australian barley used in different food products. Thus, there is a chance of azoxystrobin exposure through food to humans, and hence it is imperative to study the toxic effects of this compound. In this study, the toxic effect of azoxystrobin was evaluated to predict its adverse effects on human. Azoxystrobin at 3-30 µM (approximately 1.2-12.1 mg/L) raised the intracellular Zn2+ concentration of rat thymic lymphocytes. This increase was due to an influx of extracellular Zn2+ and a release of intracellular Zn2+. Azoxystrobin partially inhibited the temperature-dependent Zn2+ influx, thus jeopardizing the cellular Zn2+ homeostasis. Because Zn2+ is an important intracellular messenger in lymphocytes, this altered Zn2+ homeostasis might lead to adverse effects if the blood concentration of azoxystrobin reaches 3 µM or more in humans. However, by extrapolating the azoxystrobin pharmacokinetics data of rats to human, it can be predicted that such high blood concentration may be unlikely in humans.