This study investigates the influence of process conditions on the tensile properties of polyoxymethylene (POM) composites that contain three amounts of glass fiber-reinforcement (0 wt%, 15 wt% and 25 wt%). Four processing parameters - orientation of fiber, thickness of fiber layer, amount of fiber, and injection molding parameter were considered. The morphology of the fiber layer is observed by scanning electron microscopy (SEM), to elucidate the correlation between the orientation of the fibers and the mechanism of fracture of the bucking surface. The experimental results show that the maximum ultimate stress is obtained at a filling time of 1.5 s, a melt temperature of 215°C, a mold temperature of 75°C, and a packing pressure 75 MPa. SEM revealed that the composite contained two distinct layers. The fibers in the frozen layer were parallel to the melt flow, while the fiber was perpendicular to the melt flow in the core layer. The thickness of the frozen layer increased with the amount of fiber, increasing tensile strength. Additionally, fiber pullout and across-matrix cracking are the main fracture mechanisms of the frozen layer, whereas failure of the fiber-matrix interface is the major fracture mechanism in the core layer.

When a DNA probe hybridizes a DNA target and generates a G/A mismatch in the probe-target DNA heteroduplex, the mismatch enzyme, mutY, will cut the A base at the site of the mismatch. This specific cleavage at the mismatched A on the known probe will reveal the complementary DNA sequences of the targets. This study shows mismatch cleavage assays identify the complementary sequence of cryptic plasmid target in the extract of chlamydia infected cells in culture. In addition, the specific cleavage at a single base permits differentiation of two sequences with one base difference. This was shown in differentiating the subtypes of human immunodeficiency virus (HIV) type 1. The addition of amines in the assays increases the sensitivity by freeing the target for recycling. The combined assay system of high sensitivity and demonstrated specificity allows further evaluation for direct identification of pathogenic microorganisms in patient samples.

The delta F508 is the most common defect in the cystic fibrosis (CF) gene; it involves in a 3-base deletion in codon 508 and results in the loss of a phenylalanine residue at amino acid position 508. Our previous results have shown the mismatch enzyme cleavage at the mismatch of a DNA duplex in identifying a specific DNA sequence or a point mutation. The assay is simple and reliable. By manipulating the melting temperature (Tm) for the hybrids of the DNA targets and the deoxynucleotide probes, the mismatch cleavage assays are able to detect the most common defective CF gene, delta F508. The assays with a delta F508 and a normal wild-type probe can differentiate the three genotypes, i.e., delta F508/delta F508, delta F508/normal and normal/normal. Furthermore, the addition of ammonium acetate amplifier to the assay for recycling the target DNA can increase the sensitivity to a level that is sufficient to detect the mutated target in a few micrograms of genomic DNA without the aid of PCR amplification. The detection of the base deletion, the amplification of sensitivity and the differentiation among the genotypes of normal, carrier delta F508 and mutant delta F508 suggest the useful application of mismatch cleavage in genetic diagnosis at the DNA level.

We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay. In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes. In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes. The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments. Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.

A G:C --> T:A mutational hotspot at codon 249 of the p53 tumor suppressor gene has previously been identified in hepatocellular carcinoma (HCC) of patients from Qidong, China and southern Africa in which aflatoxin B1 (AFB1) and hepatitis B virus (HBV) are known synergistic risk factors. We have examined p53 mutation patterns of HCC from geographic areas in which the risk factors vary. Nine HCC lines and four hepatoblastoma lines (HB) were examined for p53 gene mutations and the relationship with HBV infection. Five of the nine HCC lines had homozygous mutation or deletion randomly distributed in exons 6-8, whereas none of the four HB cell lines had p53 mutations. One of the four HB lines (HepG2) had an N-ras mutation at codon 61 position 2. The p53 point mutations in the three HCC cell lines from Japan resulted in the amino acid changes of cysteine for tyrosine in cell line HuH 7 at codon 220 (A:T --> G:C), alanine for glycine in cell line HLF at codon 244 (G:C --> C:G), and serine for arginine in cell line HLE at codon 249 (G:C --> C:G). In addition, the deletion of 18 base pairs from codon 264 position 3 to codon 270 position 1 has resulted in the deletion of Leu-Gly-Arg-Asn-Ser-Phe from the amino acids sequences 256 - 270 in the Japanese cell line HuH 4. The cell line PLC/ PRF/5 that showed p53 mutation at codon 249 (G:C --> T:A) with substitution of serine for arginine was derived from a South African patient. Our results indicate that whereas the p53 gene is not mutated in the HB cell lines, the HCC cell lines frequently contain an abnormal p53 gene. In addition, p53 point mutations were not detected in the four Japanese HCC cell lines that were positive for genomic integration of HBV X-gene and surface antigen gene. The three Japanese HCC cell lines with p53 mutations did not contain HBV sequences, indicating that hepatocarcinogenesis associated with p53 mutation does not require the genomic integration of HBV sequences.

HUMAN hepatocellular carcinomas (HCC) from patients in Qidong, an area of high incidence in China, in which both hepatitis B virus and aflatoxin B1 are risk factors 1, were analysed for mutations in p53, a putative tumour-suppressor gene. Eight of the 16 HCC had a point mutation at the third base position of codon 249. The G --> T transversion in seven HCC DNA samples and the G --> C transversion in the other HCC are consistent with mutations caused by aflatoxin B1 in mutagenesis experiments 2,3. No mutations were found in exons 5, 6, 8 or the remainder of exon 7. These results contrast with p53 mutations previously reported in carcinomas and sarcomas of human lung, colon, oesophagus and breast; these are primarily scattered over four of the five evolutionarily conserved domains, which include codon 249 (refs 4-9). We suggest that the mutant p53 protein may be responsible for a selective clonal expansion of hepatocytes during carcinogenesis.

Five polycyclic aromatic hydrocarbons (PAHs) of different carcinogenic activities were evaluated for their effects on DNA synthesis ( 3 HTdR labeling index (L.I.)) of rat and human mammary epithelial cells (MEC) and for their effects on chromosomes in MEC‐mediated sister chromatid exchange (SCE) assays. When compared with DMSO‐treated cells, exposures of rat MEC to the two most potent carcinogens (5 μg/ml for 24 hr), i.e., 7,12‐dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B[a]P), resulted in a 45‐62% reduction in the L.I. of rat MEC. Another carcinogen, 20‐methylcholanthrene (MCA), produced a 35‐48% reduction in L.I., while the noncarcinogenic PAHs, 1,2‐benzanthracene (BA) and benzo(e)pyrene (B[e]P), showed no effect. Similarly, exposures of human MEC to DMBA and B[a]P resulted in a 50‐90% depression in L.I. while BA was significantly less effective (30% reduction). When cocultivated with Chinese hamster V‐79 cells in the presence of PAH,both rat and human MEC can activate and release the active metabolites to induce SCE in V‐79 cells. In the rat MEC‐mediated assay for all 5 PAHs, the frequencies of SCE per chromosome in DMBA‐, B[a]P‐, MCA‐, BA‐, B[e]P‐, and DMSO (solvent control)‐treated groups were 6, 3, 1.4, 0.7, 0.4, and 0.3, respectively. DMBA was most effective in increasing SCE, while B[e]P was ineffective. In the human MEC‐mediated assay, B[a]P was more effective than DMBA in inducing SCE, and the frequencies of SCE per chromosome were 4.5 and 3.6 in B[a]P‐ and DMBA‐treated groups, respectively. Comparing depression of L.I., SCE, and in vivo carcinogenecity for the 5 PAHs, SCE mediated by rat MEC is better correlated with carcinogenecity in rat than L.I. depression.

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.

One method of assessing the genotoxic effects of exposure to environmental agents is by determining the frequency of sister chromatid exchanges in exposed cells. In the present study, a procedure for observing sister chromatid exchanges in adult human bronchial epithelial cells exposed to a carcinogen has been developed. Using this procedure, the frequencies of sister chromatid exchanges in untreated cells from two individuals were found to be 5.1 +/- 1.8 and 6.5 +/- 1.4 per metaphase (mean +/- SD). Cells from the first individual exposed to 7,12-dimethylbenz[a]anthracene at 0.5, 1 and 2 mug/ml had 7.8 +/- 2.2, 13 +/- 3.1, 18.7 +/- 3.7 sister chromatid exchanges per metaphase, respectively. This method is likely to be particularly useful for observing sister chromatid exchanges in cells that have a relatively slow growth rate in the presence of bromodeoxyuridine and for which preparation of a large number of metaphase chromosomes is difficult. In addition, the procedure provides a means of assessing genotoxic effects in the lung by examining the direct effects of pollutants on the chromosomes of the target cells for human lung cancer.

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.

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 be cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites.

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.

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.

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 intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 106 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 cytochromep 450 and 536. pmol/mg protein cytochromeb 5 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.

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.

Human pulmonary macrophages were able to phagocytize BP-ferric oxide particulates in a time-dependent manner. BP was eluted from the engulfed particulates and released into the extracellular space either as free BP or as metabolites of BP. Some of the released products were mutagenic for Chinese hamster V-79 cells. A higher binding level of BP to bronchial DNA was found when bronchial explants were coincubated with PAM containing BP-ferric oxide than when incubated with BP-ferric oxide alone.

Four-month-old rhesus monkeys were injected with 65 mg of [3H] dehydroretronecine per kg body weight and sacrificed at 6,12 and 24 h following injection. By the 24th h 13% of the dose had been eliminated in the urine. Although there were no feces, the extremely high radioactivity of the bile and intestinal contents suggested this route was a major one for the excretion of this compound. The 3H was distributed throughout the body by the 6th h with the greatest percentage being in the skin and muscle. However, per gram of tissue the gastric mucosa and bile showed the highest radioactivity. Likewise, it was in the gastric mucosa that the lesions produced by dehydroretronecine were the most severe. High levels of radioactivity persisted in the gastric mucosal lysates after washings with trichloroacetic acit (TCA) while only a small percentage of the (3)H remained in the washed liver lysate. It was determined that over 20% of the 3H was bound to mucosal protein and less than 1% to the nucleic acids.