Antigenotoxic effect of Pleurotus cornucopiae extracts on the mutagenesis of Salmonella typhimurium TA98 elicited by benzo[a]pyrene and oxidative DNA lesions in V79 hamster lung cells

Pleurotus cornucopiae (PC) mushroom with a brilliant yellow pileus is found in the field and known in Japan as Tamogi dake mushroom. The purpose of this paper is to investigate the mechanism of the antimutagenic effect of PC mushroom using both the Ames test and Comet assay. We have found a strong inhibitory effect of both aqueous and organic PC extracts on the mutagenicity elicited by benzo[a]pyrene (B[a]P). This inhibition was dose-dependent in reaction mixtures containing cytosolic and microsomal fractions (S-9) from untreated rat liver as well as in those containing S-9 from aryl hydrocarbon receptor (Ah) ligand of Sudan III-treated rats. Sudan III was a potent inducer of cytochrome P450 1A (CYP1A) activity. We treated rats with Sudan III to enhance the metabolic activation of B[a]P by the S-9 fraction. To explain whether this antimutagenicity was due to the inhibition of CYP1A activity that metabolically activates B[a]P, we tested the effects of the extracts on activities of CYP1A1 and CYP1A2, represented by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD), respectively. Both aqueous and organic extracts inhibited EROD activity at all dose levels, while the inhibitory effect was only observed at high doses with regard to MROD activity. Furthermore, pre-treatment of Chinese hamster V79cells with PC extracts significantly reduced H2O2-induced-DNA damage, indicating that PC extracts provide a protective effect against oxidative DNA damage. These results indicate that whole-mushroom extracts contain compounds that may inhibit the metabolic activation of B[a]P by CYP1A1 as well as prevent oxidative DNA damage.     

Lycopene and beta-carotene ameliorate catechol estrogen-mediated DNA damage

The consumption of fruits and vegetables is associated with a reduced risk of various ailments, including cancer and cardiovascular diseases. Carotenoids, such as lycopene and beta-carotene, are natural constituents of edible plants and may protect against disease. In this study, the influence of lycopene and beta-carotene on DNA damage caused by catechol-estrogens in vitro is examined. One possible mechanism by which catechol estrogens such as 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol, which cause DNA damage in naked plasmid DNA as well as in cells, contributing to the process of carcinogenesis, is through the generation of reactive oxygen species. It was found that both carotenoids at concentrations ranging from 0.25 to 10 microM significantly inhibit strand breakage induced by 4-OHE2/copper sulphate by up to approximately 90% in plasmid DNA with beta-carotene being slightly more effective. No prooxidant or cytotoxic effects were observed at the concentrations tested. These carotenoids had a similar, though reduced effect on DNA damage as measured by the comet assay, in Chinese hamster lung fibroblasts. The results obtained show that both lycopene and beta-carotene, most probably and mainly through their potent antioxidant properties, are able to inhibit catechol-estrogen-mediated DNA damage.     

Monitoring and characterization of Magnaporthe grisea isolates with decreased sensitivity to scytalone dehydratase inhibitors

Rice blast fungus isolates were collected in Kyushu to investigate resistance to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D). In 2001, failure of control of rice blast was reported in the Saga prefecture, where MBI-Ds have been used since 1998. At that time, the distribution of resistant isolates was mainly limited to that area. However, in 2002, resistant isolates were detected in all prefectures of Kyushu. DNA fingerprinting analysis showed that the mutation causing resistance to MBI-Ds had arisen independently in each area. These data suggest that resistant isolates may occur in any area and become dominant under continuous selection pressure for MBI-Ds. Nevertheless, resistant strains can be controlled by reductase inhibitors of melanin biosynthesis (MBI-R) or commercial rice seed disinfectants.     

Effects of the essential oil cyclodextrin complexes on ruminal methane production in vitro

The effects of including essential oil cyclodextrin (CD) complexes on ruminal methane and hydrogen production, volatile fatty acid (VFA) and protozoa were studied in vitro. The essential oil CD complexes used in the present study were both αCD and βCD with cineol, eucalyptus, menthol, peppermint, thyme and wasabi. Diluted rumen fluid (60 mL) was incubated anaerobically at 38°C for 6 h with the essential oil CD complex (1–40 mg as oil). Eucalyptus‐αCD reduced methane production by 40% with 10 mg as oil along with decreasing the protozoal number and increasing total VFA and proportion of propionic acid. Both wasabi‐αCD and wasabi‐βCD reduced methane production by 85% and 97%, respectively, with 10 mg as oil, though they increased hydrogen production notably. The protozoal number was unchanged by wasabi‐βCD and decreased to 50% by wasabi‐αCD. Propionic acid was increased by both wasabi‐αCD and wasabi‐βCD. The other essential oil‐CD showed no significant effect on reducing methane production. The combined use of 20 mM of fumaric acid with wasabi‐αCD (1 mg as oil) caused about a 50% depression of methane production compared to the control without increasing hydrogen production. Fumaric acid was more effective than malic acid in reducing methane production. The addition of eucalyptus‐αCD, wasabi‐αCD or wasabi‐βCD together with organic acid to rumen fluid turned out to be effective for reducing methane production with no increase in hydrogen production.     

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

Analyses of arbutin and chlorogenic acid, the major phenolic constituents in Oriental pear

The HPLC retention time, photodiode array UV spectrum analysis, and LC/MS results indicated that arbutin and chlorogenic acid are the main phenolic constituents in Oriental pear. The two compounds exist in different organs of the Yali pear, which is one of the major cultivars of Pyrus bretschnrideri. The contents of arbutin in the leaf bud, floral bud, flower, and young fruit were 11.9, 12.4, 8.29, and 9.92 mg/g fresh weight (FW), respectively. Chlorogenic acid amounts in the same organs were 2.26, 3.22, 5.32, and 3.72 mg/g FW, respectively. During development, the concentration of the two compounds in Yali pears was the greatest in young fruit (9.92 mg/g FW of arbutin and 3.72 mg/g FW of chlorogenic acid), and then declined swiftly with fruit growth to less than 0.400 and 0.226 mg/g FW, respectively, in mature fruit. Large differences existed in the distribution of the two compounds in parts of the mature fruit of 14 Oriental pear cultivars. The greatest concentration of arbutin was found in the peel (1.20 mg/g FW), which was 3-5 times greater than that found in the core and 10-45 times greater than the level in the pulp. The concentration of chlorogenic acid in the core was greater than that in the peel. The compounds in 17 cultivars of Oriental pear, including P. bretschnrideri, Pyrus pyrifolia, Pyrus ussuriensis, and Pyrus sinkiangensis, were compared with those in 5 cultivars of Occidental pear (Pyrus communis). The mean concentration of arbutin in the Oriental pear cultivars was 0.164 mg/g FW, greater than the 0.083 mg/g FW found in the Occidental pear cultivars. The greatest arbutin content was 0.400 mg/g FW, found in the Yali pear. However, the mean concentration of chlorogenic acid in the Oriental pear was 0.163 mg/g FW, less than that found in the Occidental pear (0.309 mg/g FW).     

Quantifying lignin and holocellulose content in coniferous decayed wood using near-infrared reflectance spectroscopy

To determine the independent decomposition rates of lignin and cellulose of decayed woody debris, a technique for the rapid analysis of lignin and cellulose is required. We applied a near-infrared spectroscopy (NIRS) technique to measure the lignin and holocellulose content in decayed wood. We succeeded in creating partial least-squares (PLS) models to estimate the lignin and holocellulose content in the decayed wood of five species using NIR spectra. Although the accuracy was acceptable for the estimation of a five-species mixed model (R ² = 0.970 for lignin and R ² = 0.962 for holocellulose), it was further improved when the model was applied to each species independently. This combination of NIRS and a PLS model is a valuable tool for the determination of the lignin and holocellulose content in decayed wood. The technique is time efficient (3 min per sample) and non-hazardous (no acid treatment is required).     

Characterization of monoclonal antibodies against fowl adenovirus serotype 1 (FAV1) isolated from gizzard erosion

Six clones of monoclonal antibodies (Mabs) to fowl adenovirus (FAV) serotype 1 were produced. All Mabs reacted positively by enzyme-linked immunosorbent assay. Three Mabs recognized the putative 100-kD hexon protein and reacted to serotype 1 specifically by western blot analysis but did not react to other FAV serotypes (2, 3, 4, 5, 6, 7, and 8a). These Mabs will be useful for immunodiagnosis of FAV serotype 1 infection in chickens with gizzard erosion and in further research studies involving the genomes and proteins of FAV serotype 1.