Diphenamid metabolism in plants.

Diphenamid, a herbicide, is metabolized to N-methyl 2,2-diphenylacetamide and 2,2-diphenylacetamide by the common soil fungi Trichoderma viride and Aspergillus candidus within 48 hours. The two metabolites are more toxic than diphenamid to both tomato and barnyard-grass seedlings under sterile conditions. This finding indicates that the phytotoxic moiety is not diphenamid but one of its metabolites-probably the N-methyl derivative.

Identification of transformed liver cell colonies using concanavalin A attached to agarose beads.

A method for the in vitro identification of transformed rat liver epithelial and hepatoma cells was developed using the preferential microagglutination of concanavalin A (Con A) coupled to agarose beads (Con A:agarose) to their colonies. Con A:agarose attaches to the cell surface through a specific interaction of the Con A moiety and its receptors. The attachment is dependent on the mobility and aggregation of the Con A: receptor complex on the membrane. Agents which interfere with the interaction reduced the bead density over the colonies. For the quantitative determination of transformed colonies in a mixed-cell population, also containing untransformed cells, it is essential to compare colonies of a similar size or to use the bead density per unit area as the index. When a variety of rat liver epithelial cell lines were tested, the assay proved to be simple, reproducible, and precise. It was found that the increased attachment of Con A:agarose to cell colonies is a characteristic of transformed or malignant rat liver cells.

Lack of correlation between the response to a proliferation inhibitor and other transformation markers in a mutant liver cell line.

A rat liver factor, which has been found previously to inhibit proliferation of untransformed rat liver cell lines but not of transformed liver cell lines, did not inhibit proliferation of the chemically transformed rat liver cell line, W-8. Moreover, a temperature-sensitive mutant derived from W-8 (TS-223), which exhibits an untransformed phenotype at 39.5-41 degrees and a transformed phenotype at 36 degrees, was not affected by the liver factor at either temperature. Since the factor can be incubated at 41 degrees for 4 days without loss of activity, it would seem that the regulation of cell proliferation is not necessarily linked with the expression of other markers of transformed cells.

Specific inhibition of proliferation of nonmalignant rat hepatic cells by a factor from rat liver.

A simple quantitative assay based on the colony-forming ability of cells was used to investigate the effect of partially purified factors isolated from rat liver on the proliferation of nonmalignant and malignant rat liver epithelial cells in culture. Using this assay, which differentiates between the cytostatic and cytotoxic actions of the test materials, we found that the liver factors exerted a dose-dependent cytostatic inhibition of nonmalignant liver cells but had no inhibitory effect on malignant liver cells. However, the crude fractions showed a significant activation of the proliferation of one of the malignant cell lines tested. The inhibitory effect of this material was not due to arginase, thymidine, or thymidine-degrading enzymes. The respective inhibitory and activating effects exerted by very low concentration of the liver fractions could not be resolved by our separation methods. Even though the material may contain a mixture of activators and inhibitors, it is likely that the differential effect on the proliferative capacity of nonmalignant and malignant cells is due to the altered state of the malignant cells.

Investigation of the possible involvement of ketonic derivatives of polycyclic hydrocarbons in hydrocarbon carcinogenesis.

5,6-Dihydro-5-oxo-7,12-dimethylbenz(a)anthracene and 5,6-dihydro-6-oxo-7,12-dimethylbenz(a)anthracene are described. These compounds, which are isomeric with 7,12-dimethylbenz(a)anthracene-5,6-oxide (the K-region epoxide), were inactive in tests for tumor-initiating activity in mouse skin and tumor production in the s.c. tissue of the mouse.

Carcinogenesis by nonmutagenic chemicals: early response of rat liver cells induced by methapyrilene.

Although methapyrilene (MP) produces hepatocellular carcinomas in rats, it does not elicit many of the cellular responses induced by other hepatocarcinogens. We have investigated the early changes induced in rat liver epithelial cell cultures by MP using morphological, cytochemical, and cytofluorometric techniques. Within 2 h of MP treatment, inclusion bodies which were stainable with lipid stains were observed in the cytoplasm. Ultrastructurally, they resembled lamellar bodies with alternating light and dark lamellae. These bodies were transient in nature, since they disappeared within 24 h of removal of MP. They were, however, retained in the cytoplasm as long as MP was present in the medium. Lamellar bodies appear to be induced in the presence of histamine H1 receptor-blocking agents, since methaphenilene and diphenhydramine produced this reaction, but cimetidine, an H2 antagonist, did not. Morphologically, mitochondria of control cells were long and rod-like, whereas they were short or bizarrely shaped in the MP-treated cells. Moreover, a quantitative increase was observed in the mitochondrial content of the treated cells, when intact liver cells vitally stained with Rhodamine 123 were analyzed with a fluorescence-activated cell sorter. A significant increase in binucleated cells was observed when liver cells were exposed for 10 to 12 days with MP. Collectively, these results suggest that MP might perturb the cytoskeletal elements leading to an alteration in the nuclear and mitochondrial makeup of rat liver cells.

Biochemical basis for cytotoxicity of 7,12-dimethylbenz(a)anthracene in rat liver epithelial cells.

When the effects of 7,12-dimethylbenz(a)anthracene (DMBA) on normal and malignant rat liver epithelial cells were compared in a colony inhibition assay, this carcinogen showed a preferential cytotoxic action on the normal cells. In investigations of the biochemical basis of this selective toxicity, it was found that both cell lines were similarly effective in binding DMBA to DNA and that both cell lines had the capacity to metabolize this carcinogen. However, the hepatoma cells were more efficient than were the normal cells in generating very polar metabolites (not organic solvent extractable). These studies suggest that the basis of the resistance of the hepatoma cells to the toxicity induced by DMBA lies in their ability to detoxify biologically active metabolites. Several phenols were examined as possible toxic metabolites of DMBA, but these were not toxic at dose levels at which DMBA kills most of the normal cells.

Inability of methapyrilene to induce sister chromatid exchanges in vitro and in vivo.

The induction of sister chromatid exchanges (SCE) by the hepatocarcinogen methapyrilene hydrochloride was investigated using appropriate in vitro and in vivo mammalian cell systems. Methapyrilene, even at the maximum tolerated dose, did not induce SCE in Chinese hamster ovary cells (CHO) or when CHO cells or hamster lung fibroblasts, V-79, were cocultivated with early cultures of rat liver epithelial cells, which are known to metabolize different classes of chemical carcinogens to active forms. Moreover, a hybrid clone of cells (formed by fusion of CHO cells with rat liver epithelial cells), which is highly sensitive to SCE formation by a number of xenobiotics, failed to produce SCE after treatment with methapyrilene. Experiments in vivo with bone marrow cells and in vitro with CHO cells cocultivated with primary hepatocytes from rats also confirmed the inability of methapyrilene to induce SCE in the indicator cells. Since aflatoxin B1 induced SCE in the in vitro and in vivo models, it may be concluded that methapyrilene does not induce SCE at a concentration which is not cytotoxic to the indicator cells in the different systems described. Autoradiographic studies in cultured rat liver cells with tritiated methapyrilene showed that the label was localized in the cytoplasm but not in the interphase nuclei or in the metaphase chromosomes, indicating a lack of interaction of methapyrilene with the nuclear macromolecules of the putative target cells for methapyrilene.

Identification of transformed rat liver epithelial cells in culture.

The growth potential of normal and transformed rat liver epithelial cells in culture was investigated under various experimental conditions. The control cells failed to form colonies over a base layer of living parent cells, whereas transformed cells formed distinct colonies. Contact with living normal liver cells is required for the growth inhibition of the untransformed cells. Malignant cells capable of producing hepatocellular carcinomas in vivo exhibited signs of invasion in vitro. Altered properties of hepatic cells during the early stages of transformation can be assessed by plating such cell population over a living base layer of normal liver cells.

Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH-DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH-DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH-DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment and characterization of immortalized human cell lines from prostatic carcinoma and benign prostatic hyperplasia.

New human prostate cell lines were developed from prostatic carcinoma (BRF-41T) and BPH (BRF-55T). Primary cultures were initiated from cellular outgrowths of explanted tissues. A serum-free medium, BRFF-HPC1, was developed for growing human prostatic cancer cells. Cell strains were immortalized with pRSV-T plasmid to generate permanent cell lines that exhibited an epithelial morphology. Both cell lines expressed the epithelial cell markers, cytokeratins 8 and 18 as well as the prostatic marker, PSA, and the androgen receptor gene. They possess the H-ras, K-ras, and p53 genes. We hope that these new human prostatic cell lines will be useful as in vitro models for cancer research.

Ultrastructural morphology of three-dimensional colonies of cells derived from a hepatocellular carcinoma.

Cultured hepatocellular carcinoma cells were studied during anchorage-independent growth in semi solid medium (Methocel). The regular occurrence of mitotic figures both at the surface and within the colonies precludes the possibility of such colonies being formed by re-aggregation. The estimated population doubling time in the three-dimensional (3-D) colonies is consistent with those two-dimensional of (2-D) colonies. Structures resembling bile canaliculi were observed between the closely opposed membranes from the well packed adjacent cells. Cell surface and ultrastructural features of the colonies and individual cells are presented and comparisons made with 2-D growth of normal and malignant liver cells in vitro. The formation of 3-D colonies may not only be an assay for transformed cells but also for predicting the type of tumors produced by re-innoculation of the in vitro transformed cells.

Evidence for acrolein-modified DNA in peripheral blood leukocytes of cancer patients treated with cyclophosphamide.

Monitoring human populations for specific DNA modifications has been made possible by developing highly sensitive immunoassays employing antibodies specific for carcinogen-DNA adducts. While these techniques have been used to follow occupationally and environmentally exposed populations, results have been limited by the lack of exposure data with which to correlate adduct formation. Cancer patients treated with precisely known doses of anticancer drugs can be studied to examine the association between drug dose and adduct formation. This study examined acrolein-modified DNA in patients treated with the anticancer drug cyclophosphamide (CP) and in newly diagnosed patients prior to treatment. Employing 2 different detection methods, enzyme-linked immunosorbent assay (ELISA) and immuno-dot blot (IDB), acrolein-modified DNA was identified in a total of 6 of 12 (50%) treated patients and in 0 of 15 untreated patients. Formation of acrolein-modified DNA was examined as a function of lifetime CP dose, recent CP dose, time since last treatment, regime of treatment, and smoking history; however no clear trends were observed.

Establishment of immortalized alveolar type II epithelial cell lines from adult rats.

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the uptake of 2-deoxy-d-glucose in normal and malignant rat epithelial liver cells in culture.

The rate of uptake of 2-deoxyglucose in normal rat hepatic cells and chemically induced hepatoma cells in culture was studied. The hepatoma cells possessed a higher permeability to the sugar at all stages of culture. However, a decrease in the uptake of 2-deoxyglucose at confluency was observed in cells which exhibited density-dependent inhibition of growth, whether normal or malignant. The normal cells in mitosis showed an increased permeability to the sugar, whereas no such change was observed in the hepatoma cells. The kinetic studies of 2-deoxyglucose transport in normal and transformed rat liver showed a positive correlation between the increase in Vmax and the transformed state of the cells, whether they were transformed in vitro or in vivo. No changes in the apparent Km were found, indicating that there are no qualitative changes in the transport sites. The results suggest that an increase in the number of sites involved in glucose transport is a characteristic of chemically transformed rat liver epithelial cells.