Some Chloramphenicol (CAP) metabolites are suspected to be involved in the etiology of bone marrow aplasia in man. The objective of the present study was to investigate the cytotoxicity as well as the genotoxicity of CAP and six of its metabolites on human bone marrow cells (RiBM cells) and to compare these results with those obtained on human peripheral blood lymphocytes in order to estimate the relative sensitivity of the two types of cells. Three CAP metabolites NO-CAP, DH-CAP and NPAP inhibited 3H thymidine incorporation in RiBM cells at concentrations ranging from 2.10(-5) M to 2.10(-4) M. NO-CAP appeared as the most potent cytotoxic compound. CAP itself and NAPD presented some toxic effect at high concentration (1-2.10(-3) M). CAPG and HAP did not present any cytotoxic effect. By comparison, the response of human lymphocytes to CAP and its metabolites showed a similar pattern but DH-CAP was the most inhibitory compound. Concerning the genotoxic potential, NO-CAP and DH-CAP induced DNA single strand breaks in RiBM cells at concentrations of 1 and 2.10(-4) M with a dose response relationship. CAP and other metabolites were completely devoid of genotoxicity up to 4.10(-3) M. The results clearly showed that RiBM cells were much less susceptible to the genotoxic effect of CAP metabolites than human lymphocytes.

Chloramphenicol (CAP) is an antibiotic which has been implicated in the etiology of aplastic anemia in man. This product is also used in veterinary medicine. The medical use of chloramphenicol has been limited to cases where the drug is indispensible but veterinary use may lead to the presence of residues in the meat of treated animals and it is essential to establish acceptable levels of intake of such residues in order to protect human health. CAP is metabolized into at least 6 metabolites: nitroso-CAP (NO-CAP), formed in the liver, 3 excretion products: the glucuronide (CAP-G), the CAP base (NAPD), and an alcoholic derivative, HAP. Dehydro-CAP (DH-CAP) and the dehydro-CAP base (NPAP) are formed by enterobacteria in the large bowel. The objective of the present study was to investigate (1) the cytoxicity of CAP and its metabolites and (2) their ability to induce DNA damage in human cells. This work was performed with human peripheral blood lymphocytes (PBL) and with a lymphoma cell line (Raji).

We report here data on the spontaneous resumption of proliferation in long-term primary cultures and we show that the proliferating areas are neoplastic. Normal rat hepatocytes were explanted in serum-supplemented Ham F12 medium and maintained over 8 months without transfer. The cells remained quiescent for the first 10 weeks and they were not tumorigenic when injected into nude mice. Later, without any modification of the culture conditions or transfer, progressive changes spontaneously occurred. Foci of dividing cells were detected, some displaying gamma-glutamyl-transpeptidase (gamma-GT) activity and F-actin fragmentation. These proliferating foci overcame the quiescent population. When injected into nude mice, the 15-week-old primary cultures were highly tumorigenic, with a 3-6 week latency for tumour formation. Furthermore, a cell line was derived from a primary culture started with a liver carcinogen promoter (biliverdin-enriched medium). This cell line proliferated rapidly and differed from a liver epithelial line, also established from our primary cultures, in its 1 karyotype (hyperploidy and translocation on chromosome 3), 2 requirement for arginine to proliferate, 3 gamma-GT positive reaction correlated to changes in actin fibre pattern, 4 sensitivity to protease inhibitors (i.e. alpha2 macroglobulin, PMSF) and 5 tumorigenicity. Long-term primary cultures and the karyotypically defined cell line are useful tools for further studies on in vitro genetic deviations.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.

The organization of microfilaments and microtubules was studied in human epithelial HBL-100 cells at different steps in their malignant conversion. This conversion was obtained either spontaneously upon successive in vitro subcultures (approximately p. 70), or after superinfection of early passage non-tumorigenic cells (p. 28) with Kirsten murine sarcoma virus. Our results point out a clear relationship between the progression of cells toward malignancy, whatever the modality of malignant transformation, and severe cytoskeleton modifications. The fragmentation of F-actin fibers was the earliest event occurring at p. 46, before the appearance of tumorigenicity. In tumorigenic cells, intracytoplasmic F-actin was nearly completely fragmented, the bundle of peripheral actin was loosened and poorly stained whereas the tubulin network was more important and more largely extended throughout the cytoplasm than in non tumorigenic cells.

The induction of primary DNA damage by the non-carcinogen 4-AAF was reinvestigated in liver cells by comparison with the carcinogen 2-AAF. DNA alkaline elution showed the appearance of single-strand breaks in total liver DNA of rats 4 h after gavage with 200 mg/kg of 4-AAF. The decrease in hepatocyte viability and yield observed in these livers after collagenase perfusion indicated a cytotoxic effect of 4-AAF treatment. Viable hepatocytes isolated from 4-AAF-treated rats as well as hepatocytes from normal rats treated with 4-AAF in vitro did not present DNA single-strand breaks.

P-450 IIC7 and IIIA2 mRNAs are constitutively expressed in the hepatic tissue under developmental control. Both forms--as well as IIIA1, 90% homologous to IIIA2 mRNA--display positive modulation by phenobarbital a prototype inducer of the liver monooxygenases and a strong promoter of experimental chemical hepatocarcinogenesis. In the present work the variations in the concentration of these P-450 mRNA were studied in rats submitted to the hepatocarcinogenic protocol of Solt and Farber. We demonstrate that a decrease in the relative concentrations of P-450 IIC7 and IIIA1, 2 mRNA is set up along the tumor promotion stage. Animals--starting the experimental carcinogenic protocol at pubertal age--show a partial inhibition of the physiological expression of P-450 IIIA1,2 mRNA associated to male sex maturation. Administration of phenobarbital results in an acceleration of the pre-neoplastic process which is concomitant with an induction of P-450 IIC7 as well as IIIA1,2 at the earlier promotion stages. P-450 mRNA concentration markedly decreases as the preneoplastic process develops. While an impaired P-450 IIIA1,2 mRNA relative abundance is observed, an inversion of the modulation of P-450 IIC7 as well as of the male phenotype marker alpha-2u-globulin mRNA arises as the tumor promotion stage progresses, both mRNA becoming repressed in response to phenobarbital.

The phenotypic response of rat liver to a carcinogenic protocol involving initiation/selection and promotion with and without phenobarbital (PB) feeding was studied in pubertal and adult male rats. Considering the early presence of preneoplastic nodular areas, it appeared that pubertal rats, initiated at 6-7 weeks, presented a higher susceptibility to the protocol than adult rats initiated at 9-10 weeks. Altered liver phenotype was characterized by: (1) gamma-glutamyl-transpeptidase (GGT) and glutathione S-transferase (GST) activities; (2) the expression of two forms of cytochrome P-450; de novo PB-inducible P-450 II B 1,2 and P-450 II C 7 normally expressed in 45-day-old rats and PB-inducible, and (3) the expression of albumin and alpha-fetoprotein cDNAs. In the absence of PB, the susceptibility of pubertal rat liver to hepatocarcinogenesis was related to a special metabolic phenotype enriched in GGT and GST activities by comparison with the quasi-normal expression of both P-450s. Adult rat liver presented a less altered pattern closer to that of noninitiated rat liver. During PB promotion, the loss of PB inducibility of P-450 II C 7 in pubertal rat liver suggested that the hormonal status of the animals could interact with initiation to modulate specific gene expression. The late phase of PB promotion revealed the loss of highly differentiated functions (P-450s, albumin), whereas enzymatic markers associated with preneoplastic foci showed a persistent high expression.

Non transformed epithelial hepatic cells (established cell line and adult rat hepatocytes) treated by liver tumor promoters, phenobarbital and biliverdin, for 24 and 48 h showed a fragmentation and loss of F-actin and a depolymerisation of microtubules. This pattern closely resembles that of transformed cells which were not susceptible to the action of promoters. In liver preneoplastic nodules obtained from rats submitted to an initiation-promotion process, actin almost completely disappeared with the concomitant appearance of a characteristic enzymatic pattern rich in GGT and GST-P. Therefore, cytoskeleton of hepatic cells is a target for tumor promoters and could play a role in promotion mechanism.

Des altérations du cytosquelette concernant en particulier les microfilaments (actine F) et les microtubules (tubuline) sont fréquemment observées dans des cellules transformées.Les cellules HBL-100 sont des cellules épithéliales humaines qui ont intégré dans leur génome du DNA de virus 5V40 et qui évoluent en culture d'un état non tumorigène aux passages précoces ( ! 30e passage) vers un état tumorigène aux passages tardifs ( % 70e passage).Une lignée de cellules tumorales (T12) a été obtenue à partir d'une tumeur induite chez la souris athymique par injection de cellules HBL-100 tardives.La surinfection des cellules HBL-100 aux passages précoces par le virus Ki-MSV, porteur de l'oncogène v-Ki-ras entraine très rapidement leur transformation néoplasique.L'état de l'actine F et de la tubuline a été étudié en immunofluorescence dans les cellules HBL-100 au cours de leur progression tumorale, dans les cellules tumorales T12 et dans ' les cellules HBL-100 surinfectées par le virus Ki-MSV.Bien que porteuses d'une information génétique SV40, les cellules HBL-100 aux passages précoces (33e p.) sont non tumorigènes et

Two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 were assayed in the SOS-chromotest using PQ 37 (uvr A) and PQ 35 (uvr+) E. coli K12 strains, in the presence of S9 fraction from Aroclor-induced rats. Both compounds were able to induce the expression of SOS functions in uvr A bacteria, in the following order: Trp-P-1 less than Trp-P-2 less than aflatoxin B1, at low concentrations (less than 125 ng/assay). In this range, the induction of SOS functions was significantly decreased in the uvr+ strain. This implies that the uvr gene product plays an important role in the repair of genotoxic damage induced by Trp-P-1 and Trp-P-2. At higher concentrations (125-500 ng/assay), Trp-P-1 became more efficient in inducing SOS functions than Trp-P-2 and excision repair was less efficient than at low concentration.

Trp-P-1, a DL-tryptophan pyrolysis product, was previously known for its mutagenic and hepato-carcinogenic properties and for inducing nucleolar damage in adult rat hepatocytes in primary culture. In this paper, the effect of Trp-P-1 was investigated on DNA, RNA and protein synthesis in hepatocytes treated with 1, 5 and 10 μg/ml for 1, 2 and 4 h in order to determine the cascade of events which could occur in cells exposed to this agent. The inhibition of the 3 biosynthetic pathways was linearly dependent on the concentration of Trp-P-1 in the culture medium for each duration of treatment. For a given concentration, the degree of inhibition depended on the duration of incubation and varied according to the type of synthesis. DNA synthesis appeared as the most rapidly and strongly impaired: 16% of control values in hepatocytes treated with 10 μg/ml Trp-P-1 for 1 hour against 50.7% for RNA and 66.7% for protein synthesis. These data indicated that DNA should be the primary target of Trp-P-1 action in good agreement with the genotoxic activity of this agent. The complete recovery of RNA synthetic capability within 2 h after the removal of Trp-P-1 from the medium could however indicate that transcription was not definitely altered. By contrast the lack of protein synthesis recovery in a Trp-P-1 free medium for 24 h might signify the extended impairment of a step of translational process at the cytoplasmic level.

The mutagenic activation of tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, was studied in a Salmonella TA98/hepatocyte mutagenesis assay. Adult rat hepatocytes in primary culture were either untreated or induced by the addition of Aroclor 1254 (2 micrograms/ml) 18-20 h before the mutagenesis test which was performed at day 1 and at day 2 after the isolation of hepatocytes. The mutagenic activation of Trp-P-1 and Trp-P-2 was studied as a function of the time of incubation and of the concentration of chemical. Trp-P-1 and Trp-P-2 incubated for 20 min in the presence of untreated hepatocytes and bacteria gave rise to a weak number of revertants which doubled the level of spontaneous mutants. Aroclor-induced hepatocytes became highly competent in mutagenic activation of tryptophan pyrolysis products and the induction ratio reached 4.9 and 7.1 for Trp-P-1 and Trp-P-2, respectively, after 60 min of incubation, on day 2 of the experiment. It should be noted that the induction ratio was higher on day 2 than on day 1. When conditions were standardized, i.e. Aroclor-induced hepatocytes on day 2, final concentration of cellular protein about 1 mg/ml, 20 min of incubation, the Salmonella/hepatocyte assay produced a linear concentration-dependent mutagenic response for Trp-P-1 and Trp-P-2. By comparing the results obtained with Aroclor-induced hepatocytes and Aroclor-induced liver S9 fraction in the Salmonella test, it could be estimated that hepatocytes were 3 times less active than the S9 fraction with regard to mutagenic activation of both Trp-P-1 and Trp-P-2.

In the Ames test, after the addition of glutathione (GSH) or uridine-5'-diphosphoglucuronic acid (UD-PGA), we observed for Trp-P-1 an unchanged or a reduced mutagenicity by both the liver and intestine S9 fraction. For Trp-P-2, the same was true when we used the intestine S9 fraction. In the presence of liver S9 fraction, Trp-P-2 mutagenicity was also decreased by the addition of UDPGA but was increased by the addition of GSH. These results show that cofactors for glucuronide and GSH conjugation may alter the metabolic activation of Trp-P-1 and Trp-P-2 and consequently their mutagenicity.