Cell culture tumor promotion experiments with saccharin, phorbol myristate acetate and several common food materials.

The BALB/c-3T3 cell neoplastic transformation system was modified to examine the tumor promoting activity of a set of substances. Following initiation of the target cells with 3-methylcholanthrene, treatment of the cultures with phorbol myristate acetate (0.01 microgram/ml; 1.5 X 10(-8) M) during the remainder of the 4-week assay interval resulted in a marked increase in both spontaneous and initiated Type III transformed foci. In contrast, a similar treatment with saccharin at 20, 100 or 500 microgram/ml (0.08, 0.4 or 2.1 X 10(-3) M) did not influence the occurrence of Type III transformed foci and did not result in a promoting response. Sodium ascorbate (2.53 X 10(-3) M) and L-tryptophan (2.45 X 10(-3) M) almost completely inhibited both spontaneous and initiated Type III transformed foci. Calcium pantothenate (2.10 X 10(-3) M) exhibited a marginal promoting effect. Under the conditions of this study in which the classical tumor promoter phorbol myristate acetate was highly active in promoting Type III transformed foci, saccharin was not active as either a direct transforming or promoting agent at doses up to 5 orders of magnitude higher.

Morphological transformation of Syrian hamster embryo cells by mezerein.

Mezerein (MEZ) has been described as a weak complete tumor promoter but an effective stage II promoter in the mouse skin initiation-promotion tumor model. In this study MEZ produced a strong transformation response when tested under code in the Syrian hamster embryo (SHE) clonal morphological transformation assay. Using a standard 7-day exposure protocol designed to detect complete carcinogens, MEZ was active at non-toxic concentrations, producing a linear response between 0.3-10 ng/ml in a log-log plot of transformation activity versus concentration. These concentrations were in the same range as those which had been shown to elicit promotion activity in several in vitro cell culture systems. Our data suggest that the SHE assay has the ability to detect some tumor promoters under the same conditions used to test for complete carcinogens. The possibility that MEZ may possess in vivo carcinogenic activity cannot be excluded.

In vitro transformation of BALB/c-3T3 cells by chlorinated ethanes and ethylenes.

Eight chlorinated ethanes and 3 chlorinated ethylenes were tested in the BALB/c-3T3 cell transformation assay. Under the conditions of the assay, vinyl chloride and 1,1,1-trichloroethane induced a clear positive transformation response while 1,1,2-trichloroethane and trichloroethylene were weakly positive. Chloroethane, 1,1- and 1,2-dichloroethane, 1,1,1,2- and 1,1,2,2-tetrachloroethane, hexachloroethane and tetrachloroethylene were all negative in the assay conducted in the absence of an exogenous metabolic activation system. These results suggest that the BALB/c-3T3 cells possess capability to activate some, but not all, of the chlorinated hydrocarbons which exhibit species specificity in producing carcinogenicity in mice but not in rats.

Rat liver foci and in vitro assays to detect initiating and promoting effects of chlorinated ethanes and ethylenes.

Nine chlorinated aliphatics (CAs) were examined in a rat liver foci assay for tumor initiating and promoting activities. In this model, young adult male Osborne Mendel rats were first subjected to a partial hepatectomy, the test chemical was then administered at the maximum tolerated dose in the initiation or promotion phase in conjunction with diethylnitrosamine (DEN; 30 mg/kg b.w.) or phenobarbital (PB; 0.05 percent, w/w, in the diet), and gamma glutamyltranspeptidase (GGT) was used as a putative preneoplastic indicator. When administered in the promotion protocol after initiation with DEN, 1,1-dichloroethane, 1,1,2-trichloroethane (1,1,2-TCE), 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), tetrachloroethylene (TTCY), and hexachloroethane induced significant increases in GGT+-foci above control levels. 1,1,2,2-TTCE, TTCY, and 1,1,2-TCE also induced significant increases in GGT+-foci when administered in the promotion protocol without DEN initiation. Two variants of GGT+-foci were observed: the classical type associated with PB promotion, and the other, which was more diffuse, less intensely stained, resembling foci undergoing redifferentiation and associated with CAs. A number of CAs were also genotoxic in short-term in vitro tests. Taken together, the studies suggest that CAs may be complete carcinogens in vivo with weak initiating activity and stronger promoting activity.

Interlaboratory evaluation of the C3H/10T1/2 cell transformation assay.

The C3H/10T1/2 transformation assay was evaluated for its responsiveness and interlaboratory reproducibility. Two laboratories participated in this study and tested a series of 46 chemicals. The majority of these chemicals were tested under code. Of the 46 chemicals tested, seven were determined to be active in both laboratories, and 14 were determined to be inactive. When the total number of chemicals is adjusted for assays considered "no test" in either one or both laboratories as well as for tests of chemicals yielding positive results in only one laboratory, reproducible responses were obtained for 21/35, or 60%, of the chemicals tested.

The buccal space: a doorway for percutaneous CT-guided biopsy of the parapharyngeal region.

We performed percutaneous CT-guided needle biopsies with a transfacial approach in the diagnosis of lesions of the parapharyngeal region. Via the buccal space, core needle biopsy specimens were obtained in eight patients with known parapharyngeal abnormalities identified by CT and/or MR imaging. In all cases, sufficient tissue was obtained to provide a definitive histologic diagnosis. There were no significant complications. This approach provides a reliable method for evaluation of parapharyngeal lesions.

Caffeine-metabolizing capability of primary rat hepatocytes, Chinese hamster V79 cells and normal human fibroblasts in culture.

Caffeine is metabolized by primary rat hepatocytes in culture to metabolites similar to those found in rats in vivo, including 6-amino-5-[N-formylmethylamino]-1,3-dimethyluracil, one of the major caffeine metabolites produced in the rat. At a substrate-limiting level of caffeine, the rat hepatocytes metabolized 25-30% of the total added caffeine in 24 h. About 99% of the metabolites was found in the extracellular medium and rinse fractions, suggesting that on a volume basis, caffeine metabolites are in equilibrium inside and outside the cells. No detectable caffeine metabolites were produced in preparations of either Chinese hamster V79 cells, or normal human fibroblasts at caffeine concentrations from 5 microM to 5 mM. These results suggest that the varied biological effects induced by caffeine in Chinese hamster cells cannot be attributed to caffeine metabolites and further that the differential responses of Chinese hamster cells and normal human fibroblasts to caffeine are not due to qualitative or quantitative differences in caffeine metabolism in the two cell lines.

CT-appearance of intraluminal duodenal diverticulum. The "halo" sign.

The barium appearance of intraluminal duodenal diverticulum has been classically described as a "windsock" appearance. However, the CT-scan appearance of this abnormality has not been well documented. A case report of a patient with intraluminal duodenal diverticulum is presented. The authors believe the CT-scan findings in the patient are virtually pathognomonic for this lesion and propose the term "halo" sign be applied to this previously undescribed finding.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. VI. Enzymatic studies of two mutants unable to convert inosinic acid to adenylic acid.

Ade-H and ade-I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade-H did not have any detectable adenylosuccinate synthetase activity and ade-I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.

Characterization of a guanine-sensitive mutant defective in adenylo-succinate synthetase activity.

A contingent auxotrophic mutant of CHO-Kl cell is described. This mutant grows in minimal medium. Its growth is inhibited by the exogenous addition of guanine at levels which do not affect the wild type parent. Adenine reverses the guanine effect. This mutant does not complement ade-H (defective in adenylosuccinate synthetase) and has been denoted as ade-HG because of its guanine sensitivity. Some partial revertants of ade-H are found to be also sensitive to guanine, suggesting a close relationship between the ade-H locus and the guanine sensitivity. Studies of 14C-hypoxanthine incorporation into nucleotides indicated that ade-HG has some adenylosuccinate synthetase activity whether it is pre-exposed to guanine or not. Early de novo purine synthesis in ade-HG, however, is greatly inhibited when pre-exposed to guanine. This inhibition of purine synthesis by guanine is reversible and its recovery is facilitated by adenine.

Use of rodent hepatocytes for metabolic activation in transformation assays.

Although the evidence is not definitive, available information suggests that hepatocytes from appropriate rodent, and possibly other mammalian species, can be used to obtain metabolic activation in transformation assays that utilize cells with limited endogenous metabolic capabilities. On the basis of the available information, it appears that rat hepatocytes may be less suitable than hepatocytes from either mice or Syrian hamsters. In order to resolve these issues, it would be desirable to conduct a set of validation assays using: hepatocytes from several species, and possibly different mouse strains, a small selection of test chemicals from key chemical classes, and a reasonably facile and responsive assay system (BALB/c 3T3). This would provide the data needed to design one or more metabolic activation protocols for general use in screening transformation programs. In addition, the metabolic capacity of BALB/c 3T3 (3T3) cells to convert aniline-based amines to active carcinogens suggests that the application of genetic means to enhance the endogenous metabolic breadth of this or other cell strains, either by modulating control processes or adding genetic information, is a reasonable avenue of research.

Syrian hamster embryo cell transformation as a screening assay: sources of variability.

Our inter- and intralaboratory results showed that the SHE transformation assay is not yet sufficiently developed for a large scale screening program. The major source of variability is largely due to the low number of transformed colonies induced by test chemicals in any one experiment. Although this limitation presumably can be overcome by scoring a larger number of colonies, we feel such an approach would defeat the practical purpose of a screening assay. Of the experimental variables examined, we believe that medium supplements other than fetal calf serum and alternative detection/selection methods for transformation are two areas that need further development in order to improve the reproducibility of the SHE assay system for testing diverse chemicals. Until then, this system under defined conditions should remain a useful tool for research purposes.

Comparison of primary hepatocytes and S9 metabolic activation systems for the C3H-10T 1/2 cell transformation assay.

Three metabolic activation systems, primary rat hepatocytes, primary mouse hepatocytes and Aroclor 1254-induced rat liver S9 fraction were examined as exogenous activation systems for the C3H-10T 1/2 cell transformation assay. Under the conditions of the assay the primary mouse hepatocytes were more effective than the rat S9 fraction in mediating the transformation of C3H-10T 1/2 cells by the antineoplastic drug cyclophosphamide. However, the S9 fraction was more consistent than the mouse hepatocytes in the activation of dimethylnitrosamine. The primary rat hepatocytes were ineffective for activating either cyclophosphamide or dimethylnitrosamine in the transformation of C3H-10T 1/2 cells. The presence of mouse hepatocytes, but not the S9 fraction, inhibited transformation of C3H-10T 1/2 cells by 3-methylcholanthrene. These results demonstrate that the three systems were differentially effective in the activation of procarcinogens.

Bilateral involvement of the cerebellopontine angles by malignant melanoma metastasis: a case report.

Most tumors located at the cerebellopontine angle (CPA) are benign lesions, usually of neuroectodermal origin. This report describes a case of malignant melanoma with bilateral involvement of the CPA.

Meperidine and normeperidine levels following meperidine administration during labor. II. Fetus and neonate.

The time interval between the administration of meperidine to laboring patients and delivery may affect neonatal status, but sophisticated analytic techniques have not been used to determine the exposure of the fetus to meperidine at various drug-delivery intervals. By means of gas chromatography and mass spectrometry, the concentrations of meperidine and normeperidine (the principle metabolite of meperidine) were quantitated in the umbilical cord venous and arterial plasma at delivery and in the urine of the neonate for three days postpartum. Following 50 mg. of meperidine administered intravenously during labor, fetal exposure to meperidine was highest two to three hours after maternal medication while fetal exposure to normeperidine was highest four hours or more after medication. We conclude from this study that there is a definite but nonlinear relationship between the drug-delivery interval and the amount of meperidine and normeperidine an infant receives; and that the drug-delivery intervals resulting in maximum fetal exposure reported here correspond with those resulting in maximum neonatal depression reported by others.

Meperidine and normeperidine levels following meperidine administration during labor. I. Mother.

Because of the unavailability of sensitive analytic techniques, the pharmacokinetics of meperidine have not been clearly delineated in obstetric patients during labor. Moreover, the production of the active meperidine metabolite--normeperidine--has not been investigated. By means of gas chromatographic and mass spectrometric techniques, these characteristics of meperidine metabolism were evaluated in 23 pregnant patients in the present study. The data show that the disappearance curve and pharmacokinetic constants for meperidine are similar to those previously reported for nonpregnant subjects. In regard to normeperidine, the data indicate that it is produced within ten minutes after meperidine injection, increases rapidly for the next 20 minutes, and then slowly increases throughout labor. The results enumerate the pharmacokinetic constants of meperidine in obstetric patients and describe the appearance of normeperidine, the active meperidine metabolite, following meperidine administration during labor.