Low doses and thresholds in genotoxicity: from theories to experiments.

The absence of threshold in the action of genotoxic carcinogens was theoretically postulated more than thirty years ago, but continuously challenged for scientific and practical reasons. The direct experimental demonstration of the presence of a threshold for genotoxic damage is precluded by the insufficient sensitivity of the biological methods presently available. In the last twenty years the sensitivity of the methods for quantitative determination of the DNA adducts of the carcinogens was enormously improved, demonstrating linearity of the dose/adducts pattern over dose intervals of more than million-fold. The arguments more often advanced for the presence of a threshold for genotoxic carcinogens were mainly based on the action of intracellular scavengers, detoxification enzymes and repair systems, being able to block completely the genotoxic carcinogens at very low doses. This hypothesis is disproved by the constant presence of DNA adducts at extremely low doses of different carcinogens, whatever their chemical structure can be. On the other hand if genotoxic damage results from damage to proteins involved in cell division, like tubulin, there is a threshold dose for such genotoxic effects. The detailed knowledge of the genotoxicity mechanism is therefore needed for a sound carcinogenic risk assessment. Most of the genotoxic carcinogens, or their metabolites, damage directly the DNA. In this case the absence of threshold must be assumed, not only for theoretical reasons, but for the results of the experiments quantitatively relating DNA damage and very low doses of carcinogens. For the sake of clarity the "adjectivated" thresholds, like practical pragmatic, apparent and operational, must disappear from documents analysing the carcinogenic risk.

Divergent synergic effects in carcinogenesis initiation by simultaneous exposure to two genotoxic carcinogens.

Carcinogenesis is a complex and multistep process starting from initiation to tumor progression. Synergistic mechanisms can occur at every step of the process. The aim of this work was to provide information about the effect of chemical carcinogens which, if administered in combination, result in positive as well as negative synergistic effects. In order to evaluate whether for some carcinogens synergism occurs at the initiation step, we compared the effects of Ethylmethanesulfonate (EMS) on Benzo[a]pyrene (BP)-DNA adducts formation in the liver and lung of male Swiss mice treated for seven days by i.p. dose of EMS (1.2 mg/Kg b.w.) alone or by simultaneous administration of three doses of BP (25, 50, 100 mg/Kg b.w.) injected i.p. or the first day of treatment. A group of Swiss mice was treated by BP alone. At it was demonstrated in our laboratory that previous immunization toward BP influences the adduct levels of this carcinogen (14), the same treatments (BaP alone and BaP with EMS) were carried out in mice previously immunized toward BP. Liver and lung 1 BP-DNA adducts were detected in all the groups treated by both BP and EMS as compared to the group treated with BP alone. The EMS-BP association in non-immunized mice showed an antagonistic effect in the liver and a synergistic effect in the lung. In immunized mice a synergistic effect was obtained in both liver and lung. Moreover, the efficiency of both the synergistic and antagonistic effect, depended on BP dose of treatment. It is reasonable to draw the conclusion that simultaneous exposure to BP and EMS leads to different organ-specific and dose-dependent effects. This first preliminary result showed that the pattern of the interaction between genotoxic carcinogens is more complex that was foreseen, even at the stage of DNA adducts formation.

Identification of optimal conditions for the detection of benzo[a]pyrene-DNA adducts by enzyme-linked immunoadsorbent assays (ELISA).

The aim of the present report was to establish the optimal conditions for the detection of polycyclic aromatic hydrocarbon adducted to DNA by enzyme-linked immunoadsorbent assays (ELISA). Racemic 7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene ((+/-)-anti-BPDE) modified DNA samples were produced in vitro, by reacting (+/-)-anti-BPDE with calf thymus DNA, and in vivo in Swiss female mice by single i.p. injection of benzo[a]pyrene (B[a]P) (200 mg/kg body weight dissolved in tricaprylin). The BPDE adduct content in vitro and in liver and lung modified DNA was detected by direct and competitive ELISA using serial dilutions of the samples in unmodified calf thymus DNA, and polyclonal rabbit immunoglobulin-G elicited toward BPDE-DNA and BPDE-gelatin, both produced in our laboratory. The carcinogen-macromolecule conjugate in which adducts were sought could be used as an immunogen to produce a specific and potent antibody. Moreover, the modification level of the ELISA standards should be as close to the range as of the biological samples to correctly calculate the adducts, since different binding efficiency between antibody and BPDE-modified DNA is dependent on the BPDE modification level (33). Appropriate extraction of the in vitro modified samples is also necessary to guarantee the exact covalent modification level, eliminating noncovalently linked BPDE. Under these conditions, our results confirm that competitive ELISA is much more sensitive than the direct method, mainly because of the limitations caused by the coating of the antigen in each well (max 5 micrograms DNA/well), whereas the amount of DNA (modified or not) that can be employed for adduct detection by competitive ELISA increases 20-fold. The sensitivity obtained was 0.5 fmol B[a]P/microgramDNA (1.6 adducts/10(7) nucleotides).

Determination of serum levels of 2,6 diethylaniline in laboratory animal treated with Alachlor.

In order to estimate the environmental risk of the use of Alachlor, experiments on laboratory animals were conducted. Alachlor and 2,6 diethylaniline content in blood serum was quantified. Three groups of male ACI/T rats and C3H/FEJ mice were treated with three different doses of Alachlor. Six hours after the intraperitoneal injection the animals were bled and blood was collected by cardiac puncture. From serum obtained after blood centrifugation, A and DEA were extracted using diethyl ether. 2,6 diethylaniline and Alachlor determinations were carried out by high performance liquid chromatography (HPLC). The HPLC revealed that the metabolic capacity of 2,6 diethylaniline production from Alachlor in rats is dose-dependent; moreover, the animals can be subdivided into at least two groups, according to their Alachlor metabolic capacities. In mice the metabolic release of 2,6 diethylaniline was found to be practically complete at every dose tested.

Prediction of rodent carcinogenicity of further 30 chemicals bioassayed by the U.S. National Toxicology Program.

Recently the U.S. National Toxicology Program (NTP) sponsored a comparative exercise in which different prediction approaches (both biologically and chemically based) were challenged for their predictive abilities of rodent carcinogenicity of a common set of chemicals. The exercise enjoyed remarkable scientific success and stimulated NTP to sponsor a second challenging round of tests, inviting participants to present predictions relative to the rodent carcinogenicity of a further 30 chemicals; these are currently being tested. In this article, we present our predictions based on structure-activity relationship considerations. In our procedure, first each chemical was assigned to an activity mechanism class and then, with semiquantitative considerations, was assigned a probability carcinogenicity score, taking into account simultaneously the hypothesized action mechanism and physical chemical parameters.

Induction of humoral immunity toward 2-acetylaminofluorene in mice: modulation of DNA binding after 4 weeks dietary exposure to the carcinogen.

In order to investigate the modulatory effect of the immune response induced by recurrent carcinogen exposure, anti-2-acetylaminofluorene (anti-2-AAF) IgG were elicited in Swiss mice before subsequent carcinogen administration. The immunization schedule consisted of three weekly i.p. injections of 2-acetylaminofluorene (2-AAF)-gelatin conjugate, followed by a final immunogen injection 14 days later. At the end of treatment, the presence of specific anti-2-AAF antibodies in blood serum of all immunized animals was demonstrated. The immunization procedure did not affect liver metabolic activities, as evaluated using liver homogenates for the exogenous activation of 2-AAF to mutagen. After immunization, mice were fed 2-AAF pelleted in the diet at 50 and 150 p.p.m. for 4 weeks and killed at the end of treatment. The determination of DNA adducts by ELISA in liver and spleen of treated animals revealed significantly (P < 0.01-0.001) lower 2-AAF adduct levels in both tissues of immunized mice with respect to non-immunized animals (both naive and pretreated with the adjuvant alone). This result suggests that the specific humoral immunity elicited by repeated carcinogen exposure may be able to modulate the genotoxic effect induced by subsequent carcinogen administration.

Modulation of DNA binding in vivo by specific humoral immunological response: a novel host factor in environmental carcinogenesis?

To investigate the possible modulatory effect of the immune response induced by recurrent carcinogen exposure, a specific humoral immune response toward 2-acetylaminofluorene (2-AAF) was elicited in Swiss mice with repeated intraperitoneal injections of a 2-AAF-gelatin conjugate. The immunization procedure resulted in the production of specific anti-2-AAF antibodies in all treated animals. Groups of immunized and nonimmunized mice were subsequently fed 2-AAF pelleted in the diet at 50 and 150 ppm for 4 weeks. At the end of 2-AAF administration, animals were sacrificed and the content of 2-AAF-adducts in liver DNA was determined by enzyme-linked immunoadsorbent assay using a polyclonal rabbit antiserum. The comparison of the adducts levels in immunized and nonimmunized mice (receiving either the vehicle or the adjuvant alone during pretreatment) demonstrates a highly significant (p < 0.001) difference among groups, with far lower adduct levels in immunized animals. No significant difference in food consumption or liver metabolic activities was observed among experimental groups, suggesting the absence of external bias. The mechanism underlying the result observed is not yet clear; however, the experimental data strongly suggest that the specific immunological response induced by recurrent carcinogen exposure may exert a modulatory effect and act as a relevant host factor in chemical carcinogenesis.

Activation of 2,4-diaminotoluene to proximate carcinogens in vitro, and assay of DNA adducts.

1. 2,4-Diaminotoluene, which yields adducts with DNA in vivo, has been studied for its ability to form adducts in vitro. Metabolic activation with rat liver post-mitochondrial supernatant gave 300 adducts/10(6) nucleotides in calf thymus single-stranded DNA, under defined experimental conditions. 2. 2,4-Diaminotoluene-modified DNA and deoxyhomopolymers showed characteristic u.v. absorption spectra, exhibiting hyperchromic effects at 235 and 220 nm, and hypochromic effect at 260 nm. The difference spectra between diamine-modified and untreated DNA, or deoxyhomopolymer, were very similar to the spectrum of 2,4-diaminotoluene alone. 3. 2,4-Diaminotoluene-modified DNA was assayed by ELISA with specific monoclonal antibodies directed against diamine-DNA adducts. Reactions with poly-d(A) or poly-d(A-T) gave no spectral modification, and immunochemical analysis showed that the diamine did not bind to these polynucleotides. On the other hand, in the case of poly-d(G) or poly-d(C-G), strong immunoreactions were observed, demonstrating that the guanine base is involved in the binding of the diamine to DNA. 4. Monoclonal antibodies directed against different diamine-DNA adducts have shown that 80% of the in vitro metabolic activation involves the para amino group of the aromatic diamine.

Brief communication. on the problem of immunological detection of antigens in skeletal remains.

A detailed investigation with affinity-chromatography-purified fractions of antihemoglobin serum from rabbit shows that the hemoglobin content of human bones dating back 15 to 3,000 years may be very small. Some of the previous results (Ascenzi et al., 1985) indicating a high hemoglobin titer were +vitiated because of an unexpected cross-reactivity of bone extracts with the hemoglobin-unreactive fraction of the antiserum.

Production and characterization of monoclonal antibodies against DNA single ring diamines adducts.

The synthetic conjugate of the genotoxic compound 2,4 diaminotoluene (2,4 DAT) with gelatin (2,4 DAT-GEL) was employed to elicit specific antibodies directed against a restricted class of aromatic diamines. Using this immunogen, mouse monoclonal antibodies (MAbs) have been produced. These MAbs have been characterized and used in ELISA to detect 2,4 DAT covalently linked to biopolymers. The MAbs could bind to different synthetic 2,4 DAT-biopolymer adducts as well as to DNA from rats treated in vivo with the aromatic diamine, but they did not react with gelatin or biopolymers alone. The use of these MAbs has been investigated in order to develop a highly sensitive test to detect adducts of this genotoxic compound with nuclear DNA.

Specificity of rabbit antibodies elicited by related synthetic peptides.

Three 17-residue peptides, presenting from 65% to 70% sequence homology, and one endecapeptide, with no apparent homology with the first three, were chemically synthesized and investigated in their ability to elicit rabbit antipeptide antibodies. The complex cross reactivities of the antisera were investigated by testing the binding of the antibodies to the intact peptides, to their enzymatic fragments, and by the use of specific immunoadsorbents. Antipeptide antibodies may or may not crossreact with related "parent" peptides, this depending upon number, distribution, and localization of amino acid differences in low or high antigenicity regions of the immunogen. Related peptides may elicit antibodies that crossreact almost completely, and therefore not specific for one or the other "parent" peptide. Those antibodies may therefore be of little use for the selective recognition of closely related structures.

Immunological detection of hemoglobin in bones of ancient Roman times and of Iron and Eneolithic Ages.

Using an immunological method (immunoblot), we have established that hemoglobin (or hemoglobin fragments) can be quantitatively determined in old and ancient bones, some of them dating back 4500 years. It is shown that the total recovery decreases with time, but it is still effective in the older specimens. Thus, the immunological assay may prove useful to solve problems relevant to paleontology and paleopathology.

Monitoring of urinary mutagenicity in workers exposed to low doses of 2,4,7-trinitro-9-fluorenone.

A monitoring of the urinary mutagenicity in workers occupationally exposed to low doses of 2,4,7-trinitro-9-fluorenone (TNF) was undertaken. Urine concentrate of 22 exposed workers (11 smokers and 11 nonsmokers) and 18 presumedly unexposed workers (7 smokers and 11 nonsmokers) were assayed for mutagenicity in Salmonella typhimurium strain TA98 with the plate incorporation technique. In this test system none of the urine concentrate was effective as a mutagen, either in the absence or presence of S9. Fifteen urine samples (8 from exposed workers, 7 from referents) were also tested in the microtiter fluctuation assay. With this technique smoking habits were significantly related to urinary mutagenicity in tests performed with metabolic activation. In neither case however was the association between presumed exposure and urinary mutagenicity significant. These results were evaluated on the basis of urinary mutagenicity data obtained from rats exposed to TNF by different routes. It was shown that the observed urinary mutagenicity accounts for a minor fraction of the administered TNF dose (about 0.1 to 0.2%, depending on the route of exposure); thus it is possible that low-level exposure to TNF could escape detection by urinary mutagenicity monitoring.

Synovial sarcoma: an Australian series of 48 cases.

A series of 48 cases of synovial sarcomas submitted to the Australasian Soft Tissue Tumour Registry between 1965 and 1980 is reported. Tumours were analysed with regard to clinical features, morphology and outcome. The overall 5-yr survival rate for all assessable cases was 50%. A strong relationship between size and survival was noted with a 73% 5-yr survival rate where tumours were less than 5 cm in maximum diameter. Biphasic tumours (16 cases) appeared to have a better prognosis, with a mean survival time of 6.1 yr as compared with 4 yr for monophasic tumours (32 cases); however, the former were generally slightly smaller tumours. Tumours with less than 5 mitoses per 10 highpower fields (2.8 sq mm) had double the mean survival time of other tumours. The histological features of swirling architecture, monotonous cell type, vascular pattern, myxoid foci, collagen production, mast cell presence and calcification are recommended as cumulative factors in arriving at a diagnosis where a biphasic pattern is not apparent.