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Fresh ex vivo cultures of normal human peripheral blood monocytes, which are nonreplicative and known to possess cytochrome P-450 associated mixed-function oxidase activity, were used to assay DNA-excision repair manifested as augmented [3H]thymidine (dThd) incorporation following treatment in culture with diverse mutagenic carcinogens. Untreated monocyte cultures established from pools of 3-6 normal donors incorporated a low level of cytoplasmic [3H]dThd throughout a majority of the cells during an 18-h incubation. This background incorporation into whole cells was 80-90% inhibited by hydroxyurea (HU) at concentrations greater than 5 mM. Dose-related increases in the cumulative 18-h [3H]dThd incorporation in monocytes were observed following treatment with UV, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), mitomycin C (MMC), N-acetoxy-acetylaminofluorene (NA-AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), and dimethylnitrosamine (DMN). The presence of HU during chemical treatment and throughout this 18 h of incubation with [3H]dThd did not influence the dose-response curves obtained with UV, MMS, NA-AAF and BaP but it increased the input dose of MNNG, MMC, DMN and AFB1 required to give peak repair incorporation. When HU was added to cultures following MNNG damage no interference with repair response was observed. HU apparently influences the extent of DNA damage by direct reactivity with these chemicals or their endogenously generated metabolites rather than inhibiting DNA-repair processes. These results provide evidence that monocytes are enzymatically proficient in base and nucleotide excision pathways and have endogenous capacity to metabolize BaP, AFB1 and DMN to DNA-damaging metabolites. As such, the monocyte is a potentially useful human cell type for detecting genotoxic chemicals and studying individuality in chemical-biological interactions.

In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epilthelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis.

Susceptibility to chemically induced transformation changed as a rat embryo cell culture was passaged. For the first 35 to 60 passages, the cultures were diploid and resistant to transformation by chemical carcinogens. However, cultures infected with a murine leukemia virus were transformed by chemicals. For the next 60 passages, the cultures were heteroploid, but retained contact inhibition and were not tumorigenic. Even without addition of heterotypic viruses, these heteroploid cultures could be transformed by chemicals, but the endogenous rat C-type virus could be demonstrated in the transformed cultures. At higher passages, the rates of spontaneous transformation gradually increased so that the cultures could not be used for transformation studies. Chemically induced transformation of the stable heteroploid cell line (F1706) was manifested by an easy to read focal alteration. Initial observations based on these foci were confirmed by inoculating the morphologically altered cells into isogeneic newborn rats. A number of carcinogenic and noncarcinogenic chemical analogues were tested for their ability to transform F1706 cultures. The compounds tested included 4 azo dyes, 12 polycyclic hydrocarbons, 12 aromatic amines, and 7 miscellaneous compounds. Based on the known activities of the same chemicals in rodents, certain active compounds failed to induce transformation in any test, and others induced transformation in only some tests, but these in vitro tests, if used as a screening assay, would have been correct in 82% of all individual tests, and over-all, would have correctly predicated the carcinogenic activity of 33 of the 35 agents tested.

Latex particle agglutination for Streptococcus pneumoniae was evaluated in 76 patients. Fifteen of these patients had invasive disease due to S. pneumoniae including 12 with meningitis, 2 with occult bacteremia and 1 with suppurative arthritis. Five of the patients with meningitis also had bacteremia. Pneumococcal antigen was detected in the cerebrospinal fluid of 9 of the 12 patients with meningitis (sensitivity 75%). However, antigen was detected in the serum of only two of the six patients with bacteremia (sensitivity 33%) and was detected in the urine of none of five patients with bacteremia (sensitivity 0%). Consequently latex particle agglutination appears to be useful when cerebrospinal fluid is examined in patients with pneumococcal meningitis but does not appear to be sufficiently sensitive to warrant its use with serum or urine in patients with invasive disease due to S. pneumoniae. The specificity of the system used here appeared satisfactory, since pneumococcal antigen was not detected in any of the body fluids from the 61 patients without evidence of invasive pneumococcal disease (specificity 100%).

Seventy-five diploid human cell s-rains were subjected to a number of chemical carcinogens, including urethane and polycyclic hydrocarbons. In most cases, no visible morphological alterations were induced by any treatment. Development of morphologically altered foci was noticed in urethane-treated cultures derived from a patient with von Recklinghausen's disease. This disease is transmitted by an autosomal dominant gene, and has a high rate of spontaneous transformation of neurofibromas to neurofibrosarcomas. Attempts to isolate continuous cell lines from altered foci were successful in only two of several attempts. These continuous cell lines demonstrate altered morphology, loss of contact inhibition, accelerated growth rate, and have attained over 240 generations in a period of 140 weeks. Untreated control cultures became terminal by the 20th generation. Giemsa banding procedures showed that the chromosomal complement consisted of heteroploid human chromosomes. A second diploid cell strain derived from the above patient's sibling, also suffering from von Recklinghausen's disease, likewise was morphologically altered by urethane. Chemical transformation of human cells is difficult to induce; however, selection of genetically predisposed cells and prolonged, intermittent, and repeated chemical treatment may be important factors in achieving transformation.

The induction of lymphomas in C57BL mice by methylcholanthrene, urethan, or diethylnitrosamine was accompanied by the development of murine leukemia viral antigen in most of the lymphoid tumors. The cell-free transmission of lymphomas induced by methylcholanthrene and the development of antibody to murine leukemia virus prior to the detection of overt lymphoma in these mice suggest that unmasking of a latent leukemia virus is an indigenous actuating cause of the lymphomas.