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Biomedical subjects

I C Hsu

Publications and source records attributed to I C Hsu.

At least 37 records · Page 2Linked to original sources
PubMed1990

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes.

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.

Aflatoxin B1↗
PubMed1990

Acrylonitrile-induced sister-chromatid exchanges and DNA single-strand breaks in adult human bronchial epithelial cells.

The ability of acrylonitrile to induce cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks was studied in cultured human bronchial epithelial cells. The toxic effect as determined by cloning efficiency was observed at a dose of 600 micrograms/ml but not at doses of both 150 and 300 micrograms/ml. The frequency of sister-chromatid exchange in untreated cells was 3.7 +/- 1.3 per cell. In contrast, cells treated with acrylonitrile at 150 and 300 micrograms/ml exhibited 6.6 +/- 1.3 and 10.7 +/- 1.7 sister-chromatid exchanges per metaphase, respectively. DNA single-strand breaks were induced by acrylonitrile at dose levels of 200 and 500 micrograms/ml. The genotoxic effects on human bronchial epithelial cells that were directly exposed to acrylonitrile are of interest in relation to evidence for the higher lung cancer incidence of acrylonitrile workers in epidemiological studies.

Acrylonitrile↗
PubMed1990

Synchronous fluorescence spectroscopic, immunoaffinity chromatographic and 32P-postlabeling analysis of human placental DNA known to contain benzo[a]pyrene diol epoxide adducts.

Human placenta readily catalyzes the biotransformation of polycyclic aromatic hydrocarbons (PAHs) and other carcinogens to reactive metabolites that can damage DNA through formation of covalent adducts. Placenta is widely available for epidemiologic studies and may be a useful dosimeter for carcinogen exposures in humans. However, previous studies of human placental DNA have yielded discrepant results with respect to PAH-DNA adducts. In order to resolve some of the issues surrounding these discrepancies, placental DNA samples known to contain benzo[a]pyrene diol epoxide adducts were also analyzed by 32P-postlabeling and immunoaffinity chromatography. Results indicate that previous discrepancies can be accounted for by methodologic factors affecting the specificities of adduct assays in biological samples and suggest that human placental DNA contains adducts derived from multiple PAHs.

Benzo(a)pyrene↗
PubMed1989

Comparative effects of three carcinogens on human, rat and mouse hepatocytes.

Ultrastructural changes commonly observed in liver cells of rodents exposed to carcinogens in vivo can be induced in hepatocytes exposed to carcinogens in vitro. Human, rat and mouse hepatocytes in primary culture were treated with actinomycin D, aflatoxin B1 (AFB1) and dimethylnitrosamine (DMN). These cultured hepatocytes were examined for ultrastructural alterations following carcinogen exposure for 24 h. Similar to the effects on liver cells in vivo, the most prominent change was a segregation of the nucleolar components. Human, rat and mouse hepatocytes, dosed with 7.9 X 10(-8) M actinomycin D, developed nucleolar segregation in 86%, 98% and 55% of cells, respectively. When incubated with 3.2 X 10(-6) M AFB1, 60% of human and 84% of rat hepatocytes developed nucleolar segregation. However, exposures of mouse hepatocytes less than or equal to 3.2 X 10(-5) M of AFB1 failed to induce segregation of the nucleolus. DMN administered at a dose of 2.0 X 10(-2) M caused segregation in 11% of the rat hepatocytes and in 60% of the mouse hepatocytes. Distinct nucleolar segregation did not occur in human hepatocytes until they were exposed to a concentration of 5.0 X 10(-2) M DMN (31%). Actinomycin D, AFB1, DMN, as well as other compounds that bind to DNA and interfere with template activity cause nucleolar segregation. Morphologic changes observed in cultured rat and mouse hepatocytes correlate well with in vivo experiments with regard to the relative sensitivity of rats and mice to toxicological effects of these carcinogens. Thus, hepatocyte cultures may provide a realistic system to determine the sensitivity of human liver cells to carcinogens.

Aflatoxin B1↗
PubMed1988

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity.

DNA binding levels were determined and compared in cultured hepatocytes from male and female rats as well as other animal species following exposure to aflatoxin B1 (AFB1) or 2-acetylaminofluorene (2-AAF). When human, rat (both male and female) and mouse hepatocytes in primary culture were exposed to 2.0 X 10(-7) M [3H]AFB1 (sp. act. 2.63 microCi/nmol) for 24 h, male rat hepatocytes had the highest degree of [3H]AFB1-DNA binding (203 pmol/mg DNA) and human hepatocytes contained the next highest binding level (42 pmol/mg DNA). Hepatocytes from female rats contained 38 pmol/mg DNA while cultured mouse hepatocytes contained only 1.4 pmol/mg DNA. When the same dose of [3H]AFB1 was administered to the cultured male rat hepatocytes at 24 h, 48 h, 72 h and 1 week after seeding, and incubated for 24 h, the DNA binding levels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallel experiments to the cultured male rat hepatocytes above, the AFB1-DNA binding levels in the cultured female hepatocytes were 42, 41, 37 and 34 pmol/mg DNA respectively. Human, male and female rat hepatocytes in primary culture were exposed to 5.2 X 10(-5) M 2-acetylamino[9-14C]fluorene (sp. act. 0.0094 microCi/nmol) for 24 h. It was determined that male rat hepatocytes contained the highest amount of radioactively labeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), female rat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytes contained 0.29 nmol/mg DNA. Results from our in vitro hepatocyte culture system correlate well with in vivo animal studies dealing with species and sex differences in DNA binding and carcinogenic susceptibility. This indicates that hepatocytes in vitro maintain many of the biological properties necessary for carcinogen response similar to liver cells in vivo. In addition, comparison of genotoxic effect in cultured hepatocytes from animals as well as humans may be useful in evaluating carcinogenic potential of xenobiotics in human liver.

2-Acetylaminofluorene↗
PubMed1987

Cell and species differences in metabolic activation of chemical carcinogens.

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.

Aflatoxin B1↗
PubMed1987

Comparison of metabolic activation of carcinogens in human, rat, and hamster hepatocytes.

The activities to activate and detoxify procarcinogens were compared in intact hepatocytes from humans, Sprague-Dawley rats and Syrian golden hamsters. Mutagenic metabolites that were released from the isolated hepatocytes were detected by mutation induction in co-cultivated Salmonella typhimurium TA98. Hepatocytes from the 3 animal species all activated aflatoxin B1 (AFB1), acetylaminofluorene (AAF) and aminofluorene (AF) and released active metabolites to induce mutation in the indicator S. typhimurium T98. Hamster hepatocytes were more effective than were human and rat hepatocytes to mediate mutation of Salmonella TA98 by AFB1, AAF and AF. Hepatocytes of human and rat failed to mediate mutation by 1-aminopyrene (1-AP). Indeed, at low concentration of 1-AP and 1-nitropyrene (1-NP), the presence of the hepatocytes decreased the number of TA98 revertants. Only at higher concentrations of 1-aminopyrene and 1-nitropyrene did hamster hepatocytes increase mutation frequencies of indicator cells over the control groups. It seems that hepatocytes, particularly human hepatocytes, are better able to absorb and detoxify 1-AP and 1-NP than to activate them.

2-Acetylaminofluorene↗
PubMed1986

Comparative ultrastructural effects of aflatoxin B1 on mouse, rat, and human hepatocytes in primary culture.

Human, rat, and mouse hepatocytes in primary culture were treated with aflatoxin B1 (AFB1) and examined for ultrastructural alterations. As early as 1 h following in vitro exposure to AFB1, there were ultrastructural changes in the nuclei of rat and human hepatocytes. The most prominent change in the nuclei was a segregation of nucleolar components that resembled the segregation in liver cells of rats exposed to AFB1 in vivo. The nucleolar segregations were developed by incubating rat hepatocytes for 24 h in a medium containing as little as 0.01 micrograms of AFB1 per ml. The minimum concentration to induce the same change in human hepatocytes was 0.1 micrograms/ml. No distinct nucleolar alteration was observed in mouse hepatocytes incubated in a medium containing 10 micrograms of AFB1 per ml. Irregular nuclear chromatin condensation also developed in the cells exposed to a higher concentration of AFB1, whereas little damage was observed in mitochondria and lysosomes. The similarity in morphological changes between our in vitro model and in vivo models previously investigated indicates that the hepatocytes in primary culture maintain the biological properties necessary for carcinogen responses similar to liver cells in vivo. In addition, the morphological changes in cultured rat and mouse hepatocytes induced by AFB1 correlate with in vivo experiments insofar as mice are relatively resistant, whereas rats are sensitive to AFB1 carcinogenesis. Thus, cultured hepatocyte systems may be a valuable tool to study genetic damage which may lead to hepatocellular carcinomas in human and animal livers.

Aflatoxin B1↗
PubMed1985

Isolation and culture of hepatocytes from human liver of immediate autopsy.

Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intact lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 X 10(6) cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochrome p450 and 536 pmol/mg protein cytochrome b5 were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture.

Animals↗
PubMed1982

Studies of host factors in carcinogenesis using cultured human tissues and cells.

Recent progress in the development of conditions for culturing epithelial tissues and cells from adult humans has provided cancer researchers with an opportunity to investigate directly the various facets of carcinogenesis in human cells. Studies of activation and deactivation of several classes of chemical carcinogens have revealed that the metabolic pathways and the predominant adducts formed with DNA are generally similar in humans and experimental animals. Wide quantitative interindividual differences are found in humans and other outbred animal species. When the metabolic capabilities of specimens from different levels of biological organization are compared, the profile of benzo[a]pyrene metabolites is similar in cultured tissues and cells, but subcellular fractions, e.g., microsomes, produce a qualitatively and quantitatively aberrant pattern. To test the interactive effects of cell types in the metabolic activation of carcinogens and to assess further interindividual differences among people, human tissue- and cell-mediated mutagenesis assays have been developed. The fact that terminally differentiated cells such as pulmonary alveolar macrophages can activate benzo[a]pyrene and mediate an increase in frequencies of mutations and sister chromatid exchanges in cocultivated 'detector' cell populations, i.e., Chinese hamster V79 cells, suggests that non-target cells of chemical carcinogens may play an important role in the activation of environmental carcinogens. Malignant transformation of human epithelial and fibroblastic cells in vitro has been accomplished by several research groups. Asbestos, a cocarcinogenic agent in bronchogenic carcinogenesis, has been found to induce polyploid epithelial lesions with atypical cells in human bronchial explants. Carcinogenesis studies using cultured human tissues and cells are providing new insights into the mechanisms of carcinogenesis and are useful in identifying host factors that influence individual risk to environmental carcinogens.

Biotransformation↗
PubMed1981

Monoclonal antibody to aflatoxin B1-modified DNA detected by enzyme immunoassay.

Monoclonal antibodies were obtained after fusion of mouse P3 X 63 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin B1-adducted DNA complexed with methylated bovine serum albumin. Selected hybridomas were found to produce monoclonal antibodies specific for aflatoxin B1-modified DNA containing both the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'oxo-N5-pyrimidyl) -3-hydroxyaflatoxin B1, suggesting that these DNA adducts share a common antigenic determinant. The monoclonal antibody was not reactive towards the free aflatoxin B1-guanine adducts in solution, seven other aflatoxin derivatives, or benzo[a]pyrene-adducted DNA. A noncompetitive ultrasensitive enzyme radioimmunoassay could measure 15 fmol of aflatoxin B1-DNA adducts in 10 ng of DNA and was at least 100-fold more sensitive than the standard enzyme-linked immunosorbent assay. Competitive enzyme-linked immunosorbent assay with these monoclonal antibodies reliably quantitated aflatoxin B1 adducted in vivo to rat liver DNA at adduct levels of one aflatoxin B1 residue per 250,000 nucleotides. The competitive ultrasensitive enzyme radioimmunoassay was determined to be at least 6-fold more sensitive than the competitive enzyme-linked immunosorbent assay in analysis of aflatoxin B1-adducted DNA. Therefore, enzyme immunoassay using monoclonal antibodies will be useful analytical tools for studying both the molecular interactions of aflatoxin B1 with DNA and the occurrence of aflatoxin B1-DNA adducts in biological specimens from people exposed to this environmental carcinogen.

Aflatoxin B1↗
PubMed1981

Measurement of benzo(a)pyrene-DNA adducts by enzyme immunoassays and radioimmunoassay.

Ultrasensitive enzymatic radioimmunoassay (USERIA) was compared to radioimmunoassay and enzyme-linked immunosorbent assay in determining the amount of benzo(a)pyrene [B(a)P] metabolite covalently bound to guanine in DNA. In the USERIA approach, DNA either with or without B(a)P metabolite modification was adsorbed on the wells of microtiter plates, and rabbit antiserum to B(a)P metabolite-modified DNA [B(a)P-DNA] was then added. Antibodies reacted specifically with the B(a)P-DNA attached to the surface of the plate. After reaction between goat anti-rabbit IgG conjugated to alkaline phosphatase and rabbit IgG bound to the solid phase, this specific antigen-antibody reaction was enzymatically amplified by alkaline phosphatase conversion of [3H]adenosine 5'-monophosphate to [3H]adenosine. Following chromatographic separation from [3H]adenosine5'-monophosphate, [3H]adenosine was measured by liquid scintillation counting. The amount of [3H]-adenosine formed was linearly related to the amount of B(a)P-DNA in 10 ng DNA attached to the solid phase. As little as 3 fmol of bound B(a)P metabolite can be detected by noncompetitive USERIA, while 10 fmol of the adducts in 25 microgram DNA [1 B(a)P-DNA adduct per 7 X 10(6) nucleotides] can be measured by the competitive USERIA approach. Under our standard competitive procedure with 1 microgram DNA in the antigen-antibody reaction mixture, USERIA is approximately 500-fold more sensitive than radioimmunoassay and 5-fold more sensitive than enzyme-linked immunosorbent assay for the detection of B(a)P-DNA adducts. These highly sensitive assays should be extremely useful for studies in DNA damage and repair by B(a)P metabolites as well as in studies on environmental exposure to this ubiquitous carcinogen.

Animals↗
PubMed1980

Ultrasensitive enzymatic radioimmunoassay (USERIA) detects femtomoles of acetylaminofluorene-DNA adducts.

USERIA is a modification of radioimmunoassay and enzyme-liked immunosorbent assay. When compared to these assays, USERIA is 60- and 10-fold, respectively, more sensitive for the detection of adducts in DNA modified by the chemical carcinogen, 2-acetylaminofluorene (AAF). The specific antigen-antibody reaction is enzymatically amplified by an anti-IgG alkaline phosphatase conjugate which converts the substrate, [3H]adenosine-5'-monophosphate, to [3H]adenosine. As few as 2 fmol of AAF-DNA adducts can be detected by a competitive USERIA assay and less than 3 fmol of the specific adduct, N-(deoxyguanosine-8-yl)-AAF, can be detected using a non-competitive USERIA approach. The sensitivity of USERIA should aid efforts to measure carcinogen-DNA adducts in biological specimen samples from humans and experimental animals.

2-Acetylaminofluorene↗
PubMed1979

Relationship between sister chromatid exchange and mutagenicity, toxicity and DNA damage.

Reciprocal exchanges of DNA in sister chromatids (SCEs) are induced by various carcinogens and mutagens, although the quantitative relationship between the number of mutations and SCEs induced varies among chemicals. Nevertheless, the analysis of SCE production by various agents is often proposed as a sensitive and quantitative assay for genetic damage of the sort leading to mutation and cancer. In V-79 Chinese hamster cells we have been measuring DNA damage by alkaline elution, mutation induction as detected by 6-thioguanine resistance, and cytotoxicity as detected by colony formation for different physical and chemical agents. Some of the agents produced varying forms of DNA damage but undetectable increases in either mutation or toxicity. We report here that some undetectably mutagenic and/or toxic agents produce increases in SCE frequency and that DNA single-stranded breaks, DNA--DNA interstrand cross-links, and DNA--protein cross-links are not necessary for SCE.

Animals↗
PubMed1979

A comparison of cytotoxicity, ouabain-resistant mutation, sister-chromatid exchanges, and nascent DNA synthesis in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene.

Chinese hamster V79 cells were treated with either (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide I) or (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide II) and the nascent DNA was labeled with [Me-3H]thymidine. The cells were harvested for determination of cytotoxicity, sister-chromatid exchanges (SCE), ouabain-resistant (Or) mutations and the size of newly synthesized daughter-strand DNA. Both isomers caused dose-dependent decreases in survival of cells and in the size of nascent DNA. Increases in the frequencies of SCE and of Or mutation were found in cells treated with either isomer. However, B[a]P-diol epoxide I caused 10--20-fold more Or mutations and 50-100% more SCE than did B[a]P-diol epoxide II at equal molar dose levels. In contrast to the marked difference in the frequencies of both SCE and Or mutations caused by both compounds, the isomers induced similar reductions in the size of the nascent DNA at equal dose levels. In comparing the molecular and biological effects of the two isomers the reduction in the size of nascent DNA was more closely related to cytotoxicity than to the induction of SCE or Or mutations.

Animals↗
PubMed1979

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene.

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.

Animals↗
PubMed1979

Ultrasensitive enzymatic radioimmunoassay: application to detection of cholera toxin and rotavirus.

Rotavirus and enterotoxin-producing bacteria are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, has been developed to quantitatively detect cholera toxin and rotavirus. The method uses features of both enzyme-linked immunosorbent assay and radioimmunoassay; however, the sensitivity of the assay is 100- to 1000-fold more sensitive than the two parent assays. Ultrasensitive enzymatic radioimmunoassay should also be useful in measuring other biologically important agents such as drugs and hormones.

Alkaline Phosphatase↗