Chiho Ohta

Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length

We analyzed Hg, Zn and Cu concentrations in the liver and muscle of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan. The Hg concentration in the muscle increased proportionally with body length in the tiger sharks, whereas that in the liver increased rapidly after maturity (defined by a length of over 2.7 m). Muscle Hg levels were higher than liver concentrations in immature sharks, with the inverse trend observed in mature sharks. Notably, the Zn and Cu concentrations in the liver tended to decrease with increasing body length. This rapid increase in hepatic Hg concentration concurrent with the onset of maturity in sharks may result from the continuous intake of Hg via food and the slower growth of mature sharks. The high concentrations of the essential metals Zn and Cu in immature sharks may be explained by the physiological demands related to rapid growth.

Contamination levels of mercury in the muscle of female and male spiny dogfishes (Squalus acanthias) caught off the coast of Japan

We analyzed the total mercury (T-Hg) and stable isotopes of (13)C and (15)N in the muscle of spiny dogfish (Squalus acanthias) caught off the coast of Japan. The average body length of the female spiny dogfish sampled (94.9+/-20.2 cm, 50.5-131.0 cm, n=40) was significantly larger than that of the males sampled (77.8+/-10.8 cm, 55.5-94.0 cm, n=35), although the ages of the samples were unknown. The T-Hg concentration in the muscle samples rapidly increased after maturity in the females (larger than about 120 cm) and males (larger than about 90 cm), followed by a continued gradual increase. Contamination level of T-Hg in female muscle samples (0.387+/-0.378 microg(wet g)(-1), n=40) was slightly higher than that in male muscle samples (0.316+/-0.202 microg(wet g)(-1), n=35), probably due to the greater longevity of females. In contrast, the contamination level of T-Hg in females smaller than 94.0 cm in length (0.204+/-0.098 microg(wet g)(-1), n=20) was slightly lower than that in the males, probably due to the faster growth rate of females. Although the partial differential(13)C and partial differential(15)N values in the muscle samples increased with an increase in body length, there were no significant differences between the females (-17.2+/-0.4 per thousand and 12.4+/-0.9 per thousand, respectively) and males (-17.3+/-0.4 per thousand and 12.4+/-0.8 per thousand, respectively). A positive correlation was found between partial differential(13)C and partial differential(15)N values, suggesting trophic enrichment due to the growth.

In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450.

Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

Levels of mercury in muscle and liver of star-spotted dogfish (Mustelus manazo) from the northern region of Japan: a comparison with spiny dogfish (Squalus acanthias).

We analyzed mercury (Hg) concentrations in muscle and liver samples of star-spotted dogfish (Mustelus manazo) caught off the northern region of Japan and compared them with those of spiny dogfish (Squalus acanthias) caught in the same region. The average body length of male star-spotted dogfish specimens was significantly smaller than that of female specimens, reflecting the slower growth rate of male fish. Hg concentrations in liver and muscle increased with increases in body length and estimated age of both male and female star-spotted dogfish specimens. However, the relationships between Hg concentration in liver or muscle and body length or estimated age of male specimens differed markedly from those of female specimens, reflecting differences in growth rate and cessation of growth on reaching maturity. Marked increases in Hg concentration in liver of male and female star-spotted dogfish specimens were observed slightly later than increases in Hg concentration in muscle of those specimens due to growth cessation. These marked increases in Hg in liver may reflect increases in Hg due to the formation of mercury selenide. Similar results were previously reported in spiny dogfish specimens, except spiny dogfish showed only trace levels of Hg in liver (Endo et al., Chemosphere 77:1333–1337, 2009). The greater lipid content in liver and the larger liver size in spiny dogfish may explain the much lower levels of Hg observed in liver of spiny dogfish compared with those in the star-spotted dogfish.

Metal Concentrations in the Liver and Stable Isotope Ratios of Carbon and Nitrogen in the Muscle of Silvertip Shark (Carcharhinus albimarginatus) Culled off Ishigaki Island, Japan: Changes with Growth.

We analyzed Hg, Cd, Zn, Cu and Fe concentrations in liver samples as well as the Hg concentration and stable isotope ratios of carbon and nitrogen (δ13C and δ15N) in muscle samples from silvertip sharks (Carcharhinus albimarginatus) in Japan. Muscular and hepatic Hg concentrations increased with increased body length. However, these increases were more prominent in the liver than in the muscle samples, and appeared to occur after maturation. Hepatic Zn and Cu concentrations decreased during the growth stage, and then increased concomitantly thereafter with increases in Cd burden. Hepatic Fe concentration from males increased proportionally with increases in body length, whereas no increase was observed in samples from females, probably due to the mother-to-embryo transfer of Fe. The δ13C values tended to decrease with increases in body length, whereas no decrease in the δ15N values was observed.

In vitro metabolism of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (CB187) by liver microsomes from rats, hamsters and guinea pigs

The metabolism of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (heptaCB) (CB187) was studied using liver microsomes of rats, hamsters and guinea pigs, and the effect of cytochrome P450 (CYP) inducers, phenobarbital (PB) and 3-methylcholanthrene (MC), was also investigated. In untreated animals, guinea pig liver microsomes formed three metabolites which were deduced to be 4′-hydroxy-2,2′,3,5,5′,6-hexachlorobiphenyl (M-1), 4′-hydroxy-2,2′,3,3′,5,5′,6-heptaCB (M-2) and 4-OH-CB187 (M-3) from the comparison of GC/MS data with some synthetic authentic samples. The formation rate of M-1, M-2 and M-3 was 18.1, 36.6, 14.7 pmol h−1 mg protein−1, respectively. Liver microsomes of untreated rats and hamsters did not form CB187 metabolites. In guinea pigs, PB-treatment increased M-1 and M-2 significantly to 1.9- and 3.4-fold of untreated animals but did not affect the formation of M-3. In rats, PB-treatment resulted in the appearance of M-2 and M-3 with formation rates of 87.1 and 13.7 pmol h−1 mg protein−1, respectively, but M-1 was not observed. In hamsters, PB-treatment formed only M-2 at a rate of 29.4 pmol h−1 mg protein−1. On the other hand, MC-treatment of guinea pigs decreased the formation of M-1 and M-2 to less than 50% of untreated animals. MC-microsomes of rats and hamsters produced no metabolites. Preincubation of antiserum (300 µl) against guinea pig CYP2B18 with liver microsomes of PB-treated guinea pigs produced 80% inhibition of M-1 and the complete inhibition of M-2 and M-3. These results suggest that PB-inducible CYP forms, especially guinea pig CYP2B18, rat CYP2B1 and hamster CYP2B, are important in CB187 metabolism and that CB187 metabolism in guinea pigs may proceed via the formation of 3,4- or 3′,4′-oxide and subsequent NIH-shift or dechlorination.

Carrier-mediated uptake of nobiletin, a citrus polymethoxyflavonoid, in human intestinal Caco-2 cells

The mechanism of intestinal absorption of nobiletin (NBL) was investigated using Caco-2 cells. The uptake of NBL from the apical membranes of Caco-2 cells was rapid and temperature-dependent and the presence of metabolic inhibitors, NaN3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone, did not cause a decrease in NBL uptake. The relationship between the initial uptake of NBL and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The Km and uptake clearance (Vmax/Km) values for NBL were 50.6 and 168.1μl/mg protein/min, respectively. This clearance value was about 9-fold greater than that of the non-saturable uptake clearance (Kd: 18.5μl/mg protein/min). The presence of structurally similar compounds, such as quercetin and luteolin, competitively inhibited NBL uptake. These results suggest that uptake of NBL from the apical membranes of Caco-2 cells is mainly mediated by an energy-independent facilitated diffusion process.

Uptake of perfluorooctanoic acid by Caco-2 cells: Involvement of organic anion transporting polypeptides

The mechanism underlying the intestinal absorption of perfluorooctanoic acid (PFOA) was investigated using Caco-2 cells. The uptake of PFOA from the apical membrane of Caco-2 cells was fast, and pH, temperature, and concentration dependent, but Na+ independent. Coincubation with sulfobromophthalein (BSP), glibenclamide, estron-3-sulfate, cyclosporine A or rifamycin SV, which are typical substrates or inhibitors of organic anion transporting polypeptides (OATPs), significantly decreased the uptake of PFOA. However, coincubation with probenecid or p-aminohippuric acid, typical substrates of organic anion transporters, did not decrease the uptake of PFOA. Furthermore, coincubation with l-lactic acid or benzoic acid, substrates of monocarboxylic acid transporters, did not decrease PFOA uptake. The relationship between the initial uptake of PFOA and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The calculated Km and uptake clearance (Vmax/Km) values for PFOA were 8.3μM and 55.0μL/mg protein/min, respectively. This clearance value was about 3-fold greater than that of the non-saturable uptake clearance (Kd: 18.1μL/mg protein/min). Lineweaver-Burk plots revealed that BSP competitively inhibits the uptake of PFOA, with a Ki value of 23.1μM. These results suggest that the uptake of PFOA from the apical membranes of Caco-2 cells could be, at least in part, mediated by OATPs along with BSP.

Effect of quercetin on the uptake and efflux of aristolochic acid I from Caco-2 cell monolayers

The purpose of this study was to determine whether quercetin decreases the uptake of aristolochic acid I (AAI) from the apical membranes of Caco‐2 cells via H+‐linked MCTs at neutral pH as well as to confirm the secretion of AAI through the Caco‐2 cell monolayers via ABC transporters.

Growth-related changes in non-essential and essential metals in the liver of star-spotted smooth-hounds (dogfish) Mustelus manazo from the northern region of Japan

We analyzed Cd, Hg, Zn, Cu, Mn and Fe concentrations in liver samples from male and female star-spotted smooth-hounds at various life stages. Male sharks of this species are known to reach their maximum body length (BL) more quickly than females, while females are known to mature later and live longer than males. Hepatic Cd and Hg concentrations in males and females markedly increased after maturation, but these increases proceeded earlier in males than in females. Hepatic Zn and Cu concentrations decreased during the growth stage of males and females, and thereafter increased concomitantly with increases of Cd and Hg burdens, forming a U-shaped curve over their lifespan, and the BL at which the lowest concentrations of Zn and Cu were observed was smaller in males than in females. These gender-related differences in those metals could reflect the faster growth and earlier cessation of growth in males.