Tetsuji Nishimura

Dose‐related changes of oxidative stress and cell proliferation in kidneys of male and female F344 rats exposed to potassium bromate

It is still of importance to investigate renal carcinogenesis by potassium bromate (KBrO3), a by‐product of water disinfection by ozonation, for assessment of the risk to man. Five female F344 rats in each group were given KBrO3 at a dose of 300 mg/kg by single i.g. intubation or at a dose of 80 mg/kg by single i.p. injection, and were killed 48 h after the administration for measurements of thiobarbituric acid‐reactive substances (TBARS) and 8‐oxodeoxyguanosine (8‐oxodG) levels in the kidney. Both levels in the treated animals were significantly elevated as compared with the control values. In a second experiment, 5 male and female F344 rats in each group were administered KBrO3 at concentrations of 0, 15, 30, 60, 125, 250 and 500 ppm in the drinking water for 4 weeks. KBrO3 in the drinking water did not elevate TBARS in either sex at any of the doses examined, but 8‐oxodG formation in both sexes at 250 ppm and above was significantly higher than in the controls. Additionally, the bromodeoxyuridine‐labeling index for proximal convoluted tubules was significantly increased at 30 ppm and above in the males, and at 250 ppm and above in the females. α2u‐Globulin accumulation in the kidneys of male rats was increased with statistical significance at 125 ppm and above. These findings suggest that DNA oxidation induced by KBrO3 may occur independently of lipid peroxidation and more than 250 ppm KBrO3 in the drinking water can exert a carcinogenic effect by way of oxidative stress.

Involvement of macrophage inflammatory protein 1α (MIP1α) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

Time-dependent variation in the biodistribution of C60 in rats determined by liquid chromatography–tandem mass spectrometry

We examined the biodistribution of C(60) in rats after tail vein administration using LC-MS/MS. C(60) was detected in various tissues, such as brain, kidneys, liver, lungs, and spleen of rats. On the other hand, no C(60) was found in blood. The highest C(60) concentration was observed in the lungs, followed by spleen, liver, kidneys, and brain. These results suggested that C(60) injected in the tail vein could be filtered by lung capillary vessels and accumulate in the lungs prior to being distributed to other tissues. Moreover, C(60) not being detected in the blood indicates that clearance of C(60) from the blood by filtration might effectively occur in the lungs. The time-dependent variation in the biodistribution of C(60) was evaluated. A time-dependent decrease in C(60) concentrations was observed in all tissues, except spleen. Moreover, a decreasing trend of C(60) levels differed among tissues, which could be due to differences in accumulation. These results suggest that unmodified C(60) and/or C(60) metabolites by metabolic enzymes could be excreted into feces and/or urine. In further studies, the metabolic and excretion pathways of C(60) should be evaluated to understand the toxicokinetics of C(60).

Chronic toxicity of an environmentally relevant mixture of pharmaceuticals to three aquatic organisms (alga, daphnid, and fish)

Principles of concentration addition and independent action have been used as effective tools to predict mixture toxicity based on individual component toxicity. The authors investigated the toxicity of a pharmaceutical mixture composed of the top 10 detected active pharmaceutical ingredients (APIs) in the Tama River (Tokyo, Japan) in a relevant concentration ratio. Both individual and mixture toxicities of the 10 APIs were evaluated by 3 short-term chronic toxicity tests using the alga Pseudokirchneriella subcapitata, the daphnid Ceriodaphnia dubia, and the zebrafish Danio rerio. With the exception of clarithromycin toxicity to alga, the no-observed-effect concentration of individual APIs for each test species was dramatically higher than the highest concentration of APIs found in the environment. The mixture of 10 APIs resulted in toxicity to alga, daphnid, and fish at 6.25 times, 100 times, and 15,000 times higher concentrations, respectively, than the environmental concentrations of individual APIs. Predictions by concentration addition and independent action were nearly identical for alga, as clarithromycin was the predominant toxicant in the mixture. Both predictions described the observed mixture toxicity to alga fairly well, whereas they slightly underestimated the observed mixture toxicity in the daphnid test. In the fish embryo test, the observed toxicity fell between the predicted toxicity by concentration addition and independent action. These results suggested that the toxicity of environmentally relevant pharmaceutical mixtures could be predicted by individual toxicity using either concentration addition or independent action.

Effects of the Amino Acid Constituents of Microcystin Variants on Cytotoxicity to Primary Cultured Rat Hepatocytes

Microcystins, which are cyclic heptapeptides produced by some cyanobacterial species from algal blooms, strongly inhibit serine/threonine protein phosphatase and are known as hepatotoxins. Microcystins have many structural variations, yet insufficient information is available on the differences in the cytotoxic potentials among the structural variants. In this study, the cytotoxicities of 16 microcystin variants at concentrations of 0.03–10 μg/mL to primary cultured rat hepatocytes were determined by measuring cellular ATP content, and subsequently determined by their 50% inhibitory concentration (IC50). Differences in the amino acid constituents were associated with differences in cytotoxic potential. [d-Asp3, Z-Dhb7] microcystin-LR exhibited the strongest cytotoxicity at IC50 of 0.053 μg/mL among the microcystin variants tested. Furthermore, [d-Asp3, Z-Dhb7] microcystin-HtyR was also highly cytotoxic. These results suggest that both d-Asp and Z-Dhb residues are important in determining the cytotoxic potential of microcystin variants.

Lack of promoting effect of titanium dioxide particles on ultraviolet B-initiated skin carcinogenesis in rats.

Titanium dioxide (TiO(2)) is used in sunscreens and cosmetics as an ultraviolet light screen. TiO(2) has carcinogenic activity in the rat lung, but its effect on the skin has not been reported. We examined the promoting/carcinogenic effect of nano-size TiO(2) particles using a two-stage skin model. c-Ha-ras proto-oncogene transgenic (Hras128) rats, which are sensitive to skin carcinogenesis, and their wild-type siblings were exposed to ultraviolet B radiation on shaved back skin twice weekly for 10 weeks; then the shaved area was painted with a 100 mg/ml TiO(2) suspension twice weekly until sacrifice. All rats were killed at week 52 except for female Hras128 rats which were sacrificed at week 16 because of early mammary tumor development. Skin tumors developed in male Hras128 rats and mammary tumors developed in both sexes of Hras128 rats and in wild-type female rats, but tumor incidence was not different from controls. TiO(2) particles were detected in the upper stratum corneum but not in the underlying skin tissue layers. TiO(2) particles also did not penetrate a human epidermis model in vitro. Our data suggest that TiO(2) does not cause skin carcinogenesis, probably due to its inability to penetrate through the epidermis and reach underlying skin structures.

Cytotoxic and porphyrinogenic effects of diphenyl ethers in cultured rat hepatocytes: chlornitrofen (CNP), CNP-amino, chlomethoxyfen and bifenox.

We studied the cytotoxic and porphyrinogenic effects of four diphenyl ethers (DPEs), chlornitrofen (CNP), CNP-amino, chlomethoxyfen and bifenox, in rat hepatocytes cultured on Matrigel. Cytotoxicity was determined as a decrease in viability measured by the release of lactate dehydrogenase. Of the DPEs examined. CNP-amino was the most cytotoxic, with an LC50 value of 0.36 mM (95% confidence interval, 0.33-0.40 mM). CNP also reduced the viability in a concentration-dependent manner at the concentrations of 0.50 mM or above. In contrast, no concentration-dependent decrease in viability was observed in the chlomethoxyfen- and bifenox-treated hepatocytes at the concentrations up to 1.0 mM. To identify the enzyme involved in the metabolic activation of CNP-amino, inhibition studies were carried out using SKF 525-A (0.050 mM) and methimazole (1.0 mM). SKF 525-A, a cytochrome P450 inhibitor. quickened the onset of cell killing by CNP-amino, while methimazole, an inhibitor of flavin-containing monooxygenase (FMO), partially suppressed the cytotoxicity of CNP-amino. These results suggest that FMO plays an important role in the cytotoxicity induced by CNP-amino, while cytochrome P450 participates in the detoxification, possibly via the ring-hydroxylation. The maximum porphyrin accumulation was observed at 0.13 mM for chlomethoxyfen (18-fold) and at 0.25 mM for CNP and bifenox (17- and 21-fold, respectively). In contrast to these DPEs, the porphyrinogenic effect of CNP-amino was weak, with the maximum accumulation at 0.13 mM (at least fivefold). The predominant species was protoporphyrin IX in all of the DPE-treated cultures. These results suggest that all of the DPEs examined, possibly including CNP-amino, inhibit protoporphyrinogen oxidase, resulting in the accumulation of protoporphyrin IX.

High-temperature calcined fullerene nanowhiskers as well as long needle-like multi-wall carbon nanotubes have abilities to induce NLRP3-mediated IL-1β secretion

Because multi-wall carbon nanotubes (MWCNTs) have asbestos-like shape and size, concerns about their pathogenicity have been raised. Contaminated metals of MWCNTs may also be responsible for their toxicity. In this study, we employed high-temperature calcined fullerene nanowhiskers (HTCFNWs), which are needle-like nanofibers composed of amorphous carbon having similar sizes to MWCNTs but neither metal impurities nor tubular structures, and investigated their ability to induce production a major proinflammatory cytokine IL-1β via the Nod-like receptor pyrin domain containing 3 (NLRP3)-containing flammasome-mediated mechanism. When exposed to THP-1 macrophages, long-HTCFNW exhibited robust IL-1β production as long and needle-like MWCNTs did, but short-HTCFNW caused very small effect. IL-1β release induced by long-HTCFNW as well as by long, needle-like MWCNTs was abolished by a caspase-1 inhibitor or siRNA-knockdown of NLRP3, indicating that NLRP3-inflammasome-mediated IL-1β production by these carbon nanofibers. Our findings indicate that the needle-like shape and length, but neither metal impurities nor tubular structures of MWCNTs were critical to robust NLRP3 activation.

Cytotoxic Effects of Hydroxylated Fullerenes in Three Types of Liver Cells

Fullerenes C60 have attracted considerable attention in the biomedical field due to their interesting properties. Although there has been a concern that C60 could be metabolized to hydroxylated fullerenes (C60(OH)x) in vivo, there is little information on the effect of hydroxylated C60 on liver cells. In the present study, we evaluated the cytotoxic effects of fullerene C60 and various hydroxylated C60 derivatives, C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36, with three different types of liver cells, dRLh-84, HepG2 and primary cultured rat hepatocytes. C60, C60(OH)2 and C60(OH)36 exhibited little or no cytotoxicity in all of the cell types, while C60(OH)6–12 and C60(OH)12 induced cytotoxic effects in dRLh-84 cells, accompanied by the appearance of numerous vacuoles around the nucleus. Moreover, mitochondrial activity in liver cells was significantly inhibited by C60(OH)6–12 and C60(OH)12. These results indicate that the number of hydroxyl groups on C60(OH)x contribute to the difference of their cytotoxic potential and mitochondrial damage in liver cells.

Occurrence and behavior of the chiral anti-inflammatory drug naproxen in an aquatic environment

The present study reports on the occurrence and chiral behavior of the anti-inflammatory drug (S)-naproxen (NAP)-(S)-2-(6-methoxynaphthalen-2-yl)propionic acid-in an aquatic environment under both field and laboratory conditions. In influents and effluents of sewage treatment plants (STPs) in the Tama River basin (Tokyo), (S)-NAP was detected at concentrations of 0.03 µg L(-1) to 0.43 µg L(-1) and 0.01 µg L(-1) to 0.11 µg L(-1), respectively. The concentrations of a major metabolite, 6-O-desmethyl NAP (DM-NAP) were up to 0.47 µg L(-1) and 0.56 µg L(-1) in influents and effluents, respectively. (R)-naproxen was not detected in STP influents, although it was present in effluents, and the enantiomeric faction (= S/[S + R]) of NAP ranged from 0.88 to 0.91. Under laboratory conditions with activated sludge from STPs, rapid degradation of (S)-NAP to DM-NAP and chiral inversion of (S)-NAP to (R)-NAP were observed. During river die-away experiments, degradation and chiral inversion of NAP were extremely slow. In addition, chiral inversion of (S)-NAP to (R)-NAP was not observed during photodegradation experiments. In the river receiving STP discharge, NAP and DM-NAP concentrations reached 0.08 µg L(-1) and 0.16 µg L(-1) , respectively. The enantiomeric faction of NAP in the river ranged from 0.84 to 0.98 and remained almost unchanged with the increasing contribution of rainfall to the river water. These results suggest that the absence and decrease of (R)-NAP in river waters could indicate the inflow of untreated sewage. E