Expression of the E. coli fpg protein in CHO cells lowers endogenous oxypurine clustered damage levels and decreases accumulation of endogenous Hprt mutations.

Endogenous DNA damage clusters--two or more oxidized bases, abasic sites, or strand breaks within about 20 base pairs on opposing strands--can accumulate in unirradiated mammalian cells, and may be significant origins of spontaneous detrimental biological effects. Factors determining the levels of such endogenous clusters are largely unknown. To determine if cellular repair genotype can affect endogenous cluster levels in mammalian cells, the authors examined cluster levels, growth rates, and mutant frequencies in Chinese hamster ovary cells expressing the Escherichia coli glycosylase fpg protein, whose principal substrates are oxidized purines. In cells expressing high levels of fpg protein, the levels of oxypurine clustered damages were decreased while those of oxypyrimidine clusters and abasic clusters were unchanged. Furthermore, in these cells, the growth rates were increased and the level of spontaneous background mutants in the hypoxanthine guanine phosphoribosyl transferase gene was decreased. These results suggest that endogenous clusters are potentially detrimental DNA damages, and that their levels-as well as the detrimental consequences of their presence-can be effectively reduced by increased cellular activity of specific DNA repair proteins.

Cytotoxicity and mutagenicity of Kevlar: an in vitro evaluation.

Toxicity and mutagenicity of Kevlar 49 (PPPT; poly-para-phenylene-terephthalamide) was tested in six strains of Salmonella typhimurium (Ames test; TA97, TA98, TA100, TA102, TA1535, TA1537) with and without an external metabolic activation system (S9), as well as in a mammalian cell mutagenesis assay using V79 Chinese hamster cells. For the Ames test, liquid preincubation, which is considered particularly sensitive, was used. The cells were incubated for 24 h at a temperature of 37 degrees C either directly with Kevlar49 or with ethanol- or chloroform-extracted Kevlar49. The experiments were performed at least twice. The Ames test with six different Salmonella typhimurium strains featuring either base pair substitution or frameshift mutations revealed no cytotoxic or mutagenic activity of Kevlar49. In the mammalian cell mutagenesis assay, using 8-azaguanine (AG) as a selective agent, Kevlar49 was also devoid of cytotoxic or mutagenic activity. Both tests have to be regarded as an initial exploratory screening due to the chosen testing conditions and should be supplemented by tests at different temperatures.

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.

Production of hybrids secreting bispecific antibodies recognising CEA and doxorubicin.

A monoclonal anti-CEA secreting hybridoma (11-285-14) was made hypoxanthine, aminopterin and thymidine (HAT) sensitive by back selecting it in increasing concentrations of 8-azaguanine. Eight 8-azaguanine resistant fusion partners were selected based on growth characteristics and continued anti-CEA production. Since doxorubicin (Dox) is a hapten, it was conjugated to the carrier proteins keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) using 1-ethyl-3- (dimethyl-aminopropyl) carbodiimide. Dox-KLH and Dox-BSA conjugates were used to immunize mice and spleen cells from these mice were used for fusions with the HAT sensitive anti-CEA partner using standard hybridoma procedures. Enzyme linked immunosorbent assays (ELISAs) were developed to test the hybrids obtained for anti-CEA, anti-DOX, anti-BSA and bispecific monoclonal antibody (BsMab) activity. Sixteen fusions with spleen cells from DOX-KLH immunized mice yielded 621 hybrids of which 47 showed low level BsMab activity by ELISA. Eight fusions with spleen cells from DOX-BSA immunized mice yielded 297 hybrids. Fifty of these hybrids showing dual reactivity have been cloned and subcloned to yield 7 subclones with stable BsMab activity for CEA and doxorubicin.

Application of the standard mutagenesis assay results in underestimation of ethyl methanesulphonate-induced mutations to ouabain-resistance in Chinese hamster cells.

Chinese hamster V79 cells were exposed to ethyl methanesulphonate (EMS) and the incidence of mutant cells resistant to 8-azaguanine (8AZG), 6-thioguanine (6TG) or ouabain (OUA) was determined both by the respreading and the in situ techniques. In the former assay, the mutagen-treated cultures were grown for several days to permit the expression of mutations after which the cells were trypsinized, replated (10(5) cells/100-mm dish), and grown in medium supplemented with a selective agent. In the in situ assay, cultures were left undisturbed between EMS treatment and incubation in the presence of the selective agents. The yield of 8AZG-resistant mutants observed at optimal expression times after EMS treatment was comparable for both techniques; the induced mutation frequency (corrected for spontaneous mutation frequency) was estimated to be 82 x 10(-6) mutations per viable cell per unit dose (mM) of EMS. The frequency of 6TG-resistant mutants equalled 45 and 4 x 10(-6)/mM EMS as determined by the respreading and the in situ assays, respectively. In sharp contrast to that observed with 6TG, the frequency of OUA-resistant mutants scored by the in situ assay (30 x 10(-6)/mM EMS) proved to be an order of magnitude greater than that determined by the respreading assay (3 x 10(-6)/mM EMS). Our data therefore indicate that, when OUA is used for mutant selection, the application of the respreading technique, which has been widely adopted as the standard mammalian mutational assay over the past decade, may result in a marked underestimation of the actual mutation frequency (approximately 10-fold in V79 cells).

Mutagenicity studies on catena-(S)-[mu-[N alpha-(3-aminopropionyl)histidinato(2-)-N1,N2,O:N tau]-zinc].

Z-103 (catena-(S)-[mu-[N alpha-(3-aminopropionyl)histidinato (2-)-N1,N2,O:N tau]-zinc], CAS 107667-60-7) was examined in the bacterial mutation test, a chromosomal aberration test with mammalian cells in culture and the micronucleus test using male mice. 1. Z-103 did not increase the number of revertants in Escherichia coli WP2 uvrA when tested at up to 5000 micrograms/plate in the presence or absence of metabolic activation. And Z-103 did not increase mutants in Salmonella typhimurium SD 100 (streptomycin dependent strain) or in Salmonella typhimurium TM677 (8-azaguanine sensitive strain) when tested at up to 5000 micrograms/ml in the presence or absence of metabolic activation. 2. The chromosomal aberration test was carried out with cultured Chinese hamster lung cells (CHL). For the direct assay procedure, the cells were treated with 3.3 x 10(-4)-3.3 x 10(-6) mol/l Z-103 for 24 or 48 h, after which time chromosome preparations were made. For the metabolic activation assay procedure, the cells were treated with 1.0 x 10(-3)-3.3 x 10(-6) mol/l of Z-103 for 6 h in the presence or absence of metabolic activation, and the chromosome preparations were made after a further 18-h incubation in the absence of Z-103 and metabolic activation. Z-103 did not cause chromosome aberrations either in the presence or in the absence of metabolic activation. 3. The micronucleus test was performed in ddY male mice. Z-103 was administered orally to mice at a dose of 400, 200 or 100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

High sensitivity of Chinese hamster epithelial liver cells to toxic analogues of purines.

The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver cells are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5'-nucleotidase activity and other inhibitors. The significance of these findings is discussed.

Mutagenicity of the enantiomers of the diastereomeric bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides in bacterial and mammalian cells.

The mutagenic activities of the enantiomers of the pair of diastereomeric bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides were evaluated in histidine-dependent strains of Salmonella typhimurium and in an 8-azaguanine-sensitive Chinese hamster cell line. In strains TA 98 and TA 100 of S. typhimurium, the range in mutagenic activity observed for the four optically active isomers was less than 4- and 2-fold, respectively. The diol-epoxide with (1S,2R,3R,4S) absolute configuration and the benzylic hydroxyl group trans to the epoxide oxygen [(+)-diol epoxide-2] was the most active isomer in both strains. The enantiomeric (-)-diol-epoxide-2 isomer, with (1R,2S,3S,4R) absolute configuration identical to that of the exceptionally tumorigenic (+)-diol-epoxide-2 isomers of benzo(a)pyrene, benz(a)anthracene, and chrysene, was the least active isomer in strain TA 98 (27%) and the second most active isomer in strain TA 100 (90%). In Chinese hamster V79 cells (-)-diol-epoxide-2 was the most active of the four benzo(c)phenanthrene isomers, and a 4- to 5-fold range in mutagenic activity was observed. The differences in mutagenic activity between the four bay-region diol-epoxide isomers of benzo(c)phenanthrene in the three test systems are relatively small when compared with results from similar studies with optically active bay-region diol-epoxide isomers of three other polycyclic aromatic hydrocarbons, and may be explicable, in part, by a tendency of the hydroxyl groups of benzo(c)phenanthrene diol-epoxides to adopt comparable pseudodiequatorial conformations.

Studies in fibroblasts of patients with the Lesch-Nyhan syndrome and HPRT variants. Correlation of HPRT activity with hypoxanthine utilization and growth in selection media.

In the present study, hypoxanthine phosphoribosyltransferase (HPRT) has been investigated in fibroblasts of 19 patients from 16 different families with HPRT deficiency, concerning activity, incorporation of 14C-hypoxanthine, and growth in 8-azaguanine and HAT (hypoxanthine, azaserine, thymidine containing) selection media. According to these data we could classify the patients into 5 groups (patients with classical Lesch-Nyhan syndrome and patients with HPRT variants of types A, B, C, D). In 3 groups (patients with classical Lesch-Nyhan syndrome, HPRT variants C and D), a correlation of residual HPRT activity with the incorporation of 14C-hypoxanthine as well as growth in 8-azaguanine and HAT selection was observed. The variant A, from a patient with the classical Lesch-Nyhan syndrome, exhibited higher HPRT activity than that from all the other patients with the Lesch-Nyhan syndrome. However, the values of hypoxanthine incorporation and growth in selection media were as in the classical syndrome. The cells of variant B were resistant to azaguanine and grew in HAT selection media in the range of control cells, but had HPRT residual activities similar to those of variants A and C. For the characterization of the genetic heterogeneity of HPRT, it seems necessary to study the enzymatic properties in cell extracts as well as the purine uptake and proliferation of cells in different selection media.

DNA strand breaks in murine lymphocytes: induction by purine and pyrimidine analogues.

At the onset of culture of mouse splenic lymphocytes with concanavalin A (Con A), a 6 h pulse with 5-fluorouracil (5-FU) or 8-azaguanine (8-AG), under conditions previously shown to lead to an irreversible block of the stimulated cells in the G1 phase of the cell cycle (1,2), causes extensive DNA strand breakage. Breaks induced by the analogues early in culture were largely unrepaired even after 48 h culture. Analogues that did not block the proliferative response did not cause DNA strand breakage. Unrepaired DNA strand breaks, induced by the purine and pyrimidine analogues, provide a mechanism that can account for the block of the stimulated lymphocytes before S phase. Many strand breaks were found to exist in the DNA of normal, resting splenic lymphocytes; these were rapidly repaired within 2 h of stimulation with Con A, unlike those induced by the analogues.

Effects of metabolic inhibitors on cel lethality and mutation induction in Chinese hamster cells. II. The effect of posttreatment with non-toxic concentrations of thymidine.

The ability of posttreatment exposure to non-toxic concentrations of thymidine (TdR) to enhance the lethal effects of a number of alkylating agents, X-rays and UV and the lethal and mutagenic effects of N'-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) has been examined in V79 cell lines. TdR posttreatment enhanced the cytotoxic effects of ethyl methanesulphonate (EMS), MNU and ENU but not of UV or X-rays and increased both the spontaneous and MNU- and ENU-induced frequencies of azaguanine resistant (AZR) mutants. No significant effect of TdR on the spontaneous frequency of thioguanine resistant (TGR) mutants was demonstrated but the frequency of MNU-induced mutants to TGR premutagenic was enhanced. The effects on expression of both potentially lethal and premutagenic damage were reversed by addition of an equimolar concentration of deoxycytidine (dCdR). The enhancement in spontaneous and induced mutant frequency (IMF) at the HGPRT locus appears to be due to an alteration in the selective efficiency of urine analogous due to alteration in growth kinetics of cells exposed to TdR or treated with alkylated agents or posttreated with thymidine after alkylation damage and not to an alteration in the miscoding potential of alkylated bases.

Induction of 8-azaguanine or ouabain resistant somatic mutation of Chinese hamster lung cells by treatment with tryptophan products.

The basic fraction of tryptophan pyrolysis products (TBF) showed strong mutagenic activity on somatic cells of the lung of Chinese hamsters. In this somatic mutation test, TBF was demonstrated to have 5.6 times higher mutagenicity than diethylnitrosamine (DEN) when mutants were selected with 8-azaguanine, and 13.5-fold higher mutagenicity than DEN when mutants were selected with ouabain. From these findings, it is suggested that pyrolyzates of amino acids may have mutagenic actions on somatic cells of animals, as well as carcinogenic actions.

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts.

The cytotoxicity of the "K-region" epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several "K-region" epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ratio of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions. To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by "K-region" epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the "K-region" epoxide of benzo(a)pyrene (BP), 7,12-dimethyl-benz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine.

Low concentrations (less than or equal 20 mug/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 mug of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 mug/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; non-mutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH or US and low concentrations of AG were not mutants, but those from medium containing high concentrations (greater than or equal 30 mug/ml) of AG were mutants. Spontaneous and N-methyl-N-nitro-N-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (greater than or equal 30 mug/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH. HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.