Application of Micropore Device for Accurate, Easy, and Rapid Discrimination of Saccharomyces pastorianus from Dekkera spp.

Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.

Thioredoxin overexpression in mice, model of attenuation of oxidative stress, prevents benzene-induced hemato-lymphoid toxicity and thymic lymphoma.

OBJECTIVE: Reactive oxygen species (ROS), generated following benzene exposure, are considered to trigger the development of hematopoietic neoplasms, although little supporting evidence has been found. In this study, we examined whether the experimental elimination of ROS generated following benzene exposure prevents the development of benzene-induced hematopoietic disorders to clarify the mechanism underlying the development of benzene-induced hematopoietic disorders. METHODS: C57BL/6 mice, overexpressing human thioredoxin (h-Trx-Tg), were used to examine the possible nullification of ROS induction following benzene exposure. The experimental group was exposed to 300 ppm benzene 6 hours/day, 5 days/week, for 26 weeks, and lifetime observation followed by molecular and histopathological examinations were carried out. RESULTS: The present study using h-Trx-Tg mice showed a complete suppression of the development of thymic lymphoma induced by benzene inhalation (0% in h-Trx-Tg vs 30% in wild-type (Wt) mice). This was associated with a 48% decrease in the incidence of clastogenic micronucleated reticulocyte induction in the h-Trx-Tg mice compared with the Wt control after 2 weeks of inhalation. As underlying mechanisms, the attenuation of oxidative stress was accompanied by a complete abrogation of hemato-lymphoid toxicity, as shown by the upregulation of the activity of superoxide-dismutase, and a consequently stable ROS level, as determined by cell sorting using 2', 7'-dichlorodihydrofluorescein diacetate, along with a significant attenuation of the overexpression of a cell cycle-dependent kinase inhibitor, p21. CONCLUSION: The attenuation of benzene-induced oxidative stress and that of the consequent lymphomagenesis were observed for the first time, and these indicate a role of oxidative stress in benzene-induced clastogenesis and lymphomagenesis. (These attenuations were not seen in nonthymic lymphomas, and no leukemias developed in C57BL/6 used in this study.) During the constitutive overexpression of h-Trx, the expression of aryl-hydrocarbon receptor in h-Trx-Tg mice was downregulated, which may also contribute to the attenuation.

Etiology of bromate-induced cancer and possible modes of action-studies in Japan.

Renal cell tumors were significantly increased in male and female rats given potassium bromate at 250 and 500 mg/L in drinking water. In at least one other study renal cell tumors were produced in male rats at 125 mg/L. Among male mice given 750 mg/L of potassium bromate, there were no significant differences in renal cell tumors between treated and control groups after 88 weeks on test. In oxidative DNA damage tests 8-oxodeoxyguanosine (8-oxodG also referred to as 8-OH-dG) was induced in DNA in the male rat kidney in 1 week, and in females after 3 weeks at 500 mg/L, and also in both male and female rats at 250 mg/L, but not at 125 mg/L. DNA adducts are considered to be an initial step in the carcinogenesis process, however, the administered doses are not always sufficient to cause mutations, possibly due to DNA repair. In the two-step rat renal carcinogenesis model using N-ethyl-N-hydroxyethylnitrosamine (EHEN) as initiator, promotion activity by potassium bromate was measured using the BrdU labeling index. The promoting activity of bromate in male rats was much greater and extended to doses as low as 60 mg/L in male rats, whereas in females the response was limited to 250 and 500 mg/L. Therefore, it was concluded that the mechanisms contributing to cancer in the male rat were more complex than in the female rat. The accumulation of alpha2mu-globulin in the kidneys of male rats exposed to potassium bromate probably accounts for the greater labeling index in the male rat relative to the female rat. Accumulation of alpha(2mu)-globulin as a result of treatment with chemicals is unique to the male rat and does contribute to carcinogenic responses. Neither humans nor female rats display this response. Nevertheless, bromate must be considered carcinogenic because of the response of the female rats. The better correlation between 8-oxodG formation and tumor response indicates that dose-response information from the female rat would be much more relevant to human risk assessment. The fact that an elevation of BrdU-LI in the kidney of the female rat is consistent with the possibility that cell proliferation observed in female rats resulted from oxidative stress and/or cytotoxic responses in the kidney. Therefore, oxidative stress is most likely the mechanism of interest for cancer risk in humans.

Nine-week detection of six genotoxic lung carcinogens using the rasH2/BHT mouse model.

A 9-week in vivo rasH2/butylhydroxytoluene (BHT) model for the detection of genotoxic lung carcinogens was validated, using six potent positive test compounds, dimethylnitrosamine (DMN; 15 mg/kg, i.p.), diethylnitrosamine (DEN; 100 mg/kg, i.p.), ethylnitrosourea (ENU; 120 mg/kg, i.p.), 3-methylcholanthrene (MC; 100 mg/kg, i.p.), 7,12-dimethylbenz(a)anthracene (DMBA; 5 mg/kg, i.g.) and benzo(a)pyrene (B(a)P; 80 mg/kg, i.p.), each given to rasH2 mice of both genders by single administration for initiation followed by promoter BHT treatment. Statistically significant increase in the incidence and multiplicity of lung tumors was observed in rasH2 mice treated with BHT following exposure to all of the carcinogens tested. The data overall suggest the rasH2/BHT model to be a powerful screening tool for genotoxic lung carcinogens.

Evaluation of nonthreshold leukemogenic response to methyl nitrosourea in p53-deficient C3H/He mice.

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of "practical" thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a "practical" threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold.

Induction of different types of uterine adenocarcinomas in Donryu rats due to neonatal exposure to high-dose p-t-octylphenol for different periods.

Inappropriate exposure to estrogens in the fetal and/or newborn period can exert irreversible influence, including carcinogenesis on the reproductive system in mammals. The present study was conducted to investigate uterine carcinogenesis in Donryu rats treated neonatally with a high-dose estrogenic compound, p-t-octylphenol (OP) for different exposure periods. Female Donryu rats were subcutaneously administered 100 mg/kg/day OP every other day for the first 5 postnatal days (PNDs 1-5) or the first 2 weeks (PNDs 1-15). They received a single injection of 20 mg/kg N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) into a uterine horn at 11 weeks of age and were examined until 15 months of age. PNDs 1-5 OP-treated rats showed normal development of the female reproductive system, including uterine gland genesis and normal estrous cycling after vaginal opening. The treatment, however, accelerated an earlier occurrence of persistent estrus and increased the number of well differentiated uterine adenocarcinomas as compared with controls. This indicated that PNDs 1-5 OP treatment acts as a delayed modulator of the hypothalamus-pituitary-ovarian hormonal control system and the modulation increased the serum estrogen:progesterone ratio, resulting in induction of uterine tumors. On the contrary, PNDs 1-15 OP treatment demonstrated immediate and irreversible influences on the control system, called 'androgenization', and induced abnormal uterine development manifested by prolonged persistent estrus immediately after vaginal opening and also suppression of uterine gland genesis. In addition, uterine tumor malignancy in morphological and biological property clearly increased in this group although the total number of adenocarcinomas was not increased. The present study provides evidence that neonatal exposure to a high-dose OP enhances uterine carcinogenesis in rats, and the type of uterine tumors is changed by the periods of neonatal exposure to OP, suggesting that the mechanism of uterine tumor development is dependent upon neonatal exposure periods.

Promotion, but not progression, effects of tamoxifen on uterine carcinogenesis in mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine.

Effects of tamoxifen (TAM) on development of uterine endometrial carcinogenesis were studied in intact and ovariectomized (OVX) mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). In experiment I, animals were implanted with cholesterol (ChL, controls) or TAM (5% w/w) and/or 17beta-oestradiol (E(2), 0.5% w/w) pellets s.c. from 9 to 25 weeks of age, until the termination of the experiment, and all received a single intra-uterine administration of ENNG (12.5 mg/kg) at 10 weeks of age. They were divided into four groups: ENNG + ChL (control), ENNG + TAM, ENNG + E(2) and ENNG + TAM + E(2). Endometrial proliferative lesions (hyperplasias and/or carcinomas) were observed in all groups, the incidences in the TAM- and/or E(2)-treated groups being two times higher than in the ChL-treated control animals. High induction (11/20, 55%) of adenocarcinomas was observed in the E(2) group but this was significantly decreased in combination with TAM (2/20, 10%), no carcinomas being found in the TAM group. In experiment II, animals pre-treated with TAM (10 weeks) and receiving E(2) post-treated (4 weeks) developed adenocarcinomas, although no cancers were observed in mice treated by ChL instead of TAM. In animals pre-treated with TAM and post-treated with ChL or TAM, no adenocarcinomas were also developed. In OVX mice (experiment III), proliferative lesions were observed in the TAM- and/or E(2)-treated groups, at incidences significantly higher than in ChL-treated animals, in which these lesions were completely absent. However, no adenocarcinomas were found, only slight hyperplasias being observed in the TAM group, although the incidence of adenocarcinoma was highest in the E(2) alone group, and significantly decreased in combination with TAM, as in experiment I. These results indicate that TAM may itself exert promotion effects, while exhibiting an anti-progression influence on uterine carcinogenesis in adult mice initiated by ENNG and receiving E(2).

Neonatal exposure to p-tert-octylphenol causes abnormal expression of estrogen receptor alpha and subsequent alteration of cell proliferating activity in the developing Donryu rat uterus.

In the present study, we investigated immunohistochemically the time-course alterations in estrogen receptor alpha (ER) expression and cell proliferating activity in the developing uteri of Donryu rats exposed neonatally to a high dose p-tert-octylphenol (OP), an endocrine disrupting chemical (EDC). OP-treatment (sc injections of 100 mg/kg, every other day from postnatal days 1 to 15) induced an early and enhanced ER expression in the luminal epithelium compared with age-matched controls from postnatal day (PND) 10, and increased proliferating cell nuclear antigen (PCNA) positive cells up to PND21. At PND28, ER expression in the luminal epithelium of the OP-treated group was decreased, in association with decline in the luminal epithelial areas. PND14, the second week of life, is coincident with the normal time for differentiation when the luminal epithelium invaginates into the stroma to form uterine glands. OP-treatment, however, delayed and inhibited gland-formation, and suppressed ER expression in the invaginated-luminal and glandular epithelium at this time. These results indicate that ER expression in these sites is strongly linked with cell proliferating activity. In stromal cells, ER was expressed from PND6 in both groups without any PCNA positive cells, but significantly lower values were noted in the OP-treated group up to PND10. Our immunohistochemical investigation did not reveal any abnormalities in expression of the proto-oncogene c-fos, mitotic inhibitor p21, or epidermal growth factor antigen, although the apoptotic index in the luminal epithelium was slightly increased in the OP-treated group. These results demonstrate neonatal effects of a high dose of OP, already detectable at PND10, with early and enhanced ER expression, resulting in increase of cell proliferative activity in the luminal epithelium, though expression in the glandular epithelium was suppressed in relation to inhibited gland-genesis. The present study thus suggests that neonatal exposure to high doses of EDCs with estrogenic activity can induce abnormal differentiation in the developing rat uteri via abnormal ER expression and subsequent alteration of cell proliferating activity.

Uterine adenocarcinoma in N-ethyl-N'-nitro-N-nitrosoguanidine-treated rats with high-dose exposure to p-tert-octylphenol during adulthood.

Since many risk factors are associated with the development of uterine adenocarcinomas in humans, the etiology is unclear in most cases, although it has been pointed out that estrogen may play essential roles. To clarify the effects of exposure to p-tert-octylphenol (OP), an environmental xenoestrogen, on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 11 weeks of age and exposed thereafter to 100 mg / kg OP by s.c. injection until 15 months of age. Adult ovariectomized (OVX) rats were also treated in a similar way. OP had no effect on occurrence of persistent estrus in middle age, although uterotrophic effects were obvious in OVX rats. At the termination, development of uterine adenocarcinomas was significantly increased in animals exposed to OP during adulthood. No tumors, but a few focal hyperplasias, developed in OVX rats. These findings suggest that OP has tumor-promoting effects on ENNG-treated endometrium of rats, possibly due to direct action on the uterus, as indicated by the uterotrophic effect when a high dose of OP was given. The results provide clues to the mechanisms of influence of hormonal disrupters on uterine carcinogenesis.

Reactive Oxygen and Nitrogen Oxide Species-induced Stress, a Major Intrinsic Factor Involved in Carcinogenic Processes and a Possible Target for Cancer Prevention.

Reactive oxygen and nitrogen oxide species and their inducing stress are involved in a variety of physiological and pathological phenomena in aerobes, including humans. For multistage carcinogenic processes, reactive oxygen and nitrogen oxide species-induced stress (RONOSS) serves as a major intrinsic factor and is involved in every step. This means that free radicals, RONOSS and their inducing downstream events may be targets for cancer prevention. It is therefore of importance to elucidate the mechanisms underlying the participation of RONOSS in carcinogenesis and to apply the obtained results for establishment of strategies to control cancer development. Despite the large body of accumulated knowledge due to worldwide efforts dealing with this research field, there still remain numerous uncertainties. In this mini-review, we introduce two examples of such efforts, one concerning a renal carcinogen KBrO(3) and the other dealing with hepatocarcinogenesis caused by a choline-deficient, L-amino acid-defined diet, in order to give some idea about the current understanding of the roles of RONOSS in carcinogenesis.

The mouse rasH2/BHT model as an in vivo rapid assay for lung carcinogens.

We have demonstrated the utility of a 9-week in vivo two-stage assay for lung cancer initiating agents, using transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) and butylhydroxytoluene (BHT) as a potent lung promoter (rasH2/BHT model). In the present study, to ascertain appropriate conditions for BHT administration in this model, the effects of exposure on proliferation of alveolar type II cells in male rasH2 mice were examined. Additionally, use of BHT was validated for promotion of urethane (UR) carcinogenesis in male and female rasH2 mice. In a time-course study of a single intragastric administration of BHT at a dose of 400 mg/kg, increased bromodeoxyuridine-labeling index (LI) reached a maximum 3 days after treatment and was still observed after 7 days. In a dose-response study, effects were dose-dependent, the dose of 400 mg/kg causing eight-fold elevation as compared to the control. With repeated administration, whereas the LI was increased dramatically at first, effects gradually diminished with further exposure, and finally six BHT treatments failed to induce cell proliferation. In a two-stage model using UR as the initiator, although up to five consecutive doses of BHT were able to exert continued enhancing effects in terms of adenoma yield, no increment was evident with further treatments. The data overall indicate that a rasH2/BHT model with five weekly administrations of BHT at a dose of 400 mg/kg is most efficacious.