A number of heterocyclic amines (HCAs) have been isolated from the charred parts of broiled fish and meat, and found to be bacterial mutagens in the presence of microsomal enzymes (1, 2). HCAs also show clastogenic and mutagenic potentials in cultured mammalian cells, and are carcinogenic in rodents (2, 3). However, since the daily doses administered to rodents are extraordinarily high, compared with those to which humans are chronically exposed, extrapolation of the actual cancer risk to humans may be difficult.

The HCAs 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) are potent rodent carcinogens (4). Carcinogenic effects of these compounds are elicited from metabolic activation by the cytochrome P-450 enzyme system to reactive metabolites (5). In the absence of bioactivation, Trp-P-1 has also been shown to interact with DNA and to induce chromosomal aberrations in Chinese hamster ovary cells, indicating that both the parent compound and metabolite can cause DNA damage (6, 7). DNA damage triggers not only mutational events, which may lead to carcinogenesis, but also apoptosis or necrosis.

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is a DNA repair enzyme that is activated in the presence of DNA strand breaks, with the resultant poly(ADP-ribosylation) of proteins, such as histones, and autoacceptor sites on PARP-1 itself. Under conditions of excessive DNA damage, caspase-3 cleaves PARP-1 in cells undergoing apoptosis. However, 3-aminobenzamide, a PARP-1 inhibitor, has been shown to suppress the mitochondrial release of apoptosis inducing factor, which suggests that PARP-1 may play a critical role in non-caspase dependent apoptotic pathways as well (8). Recently, inhibition of PARP-1 by intact Trp-P-1 was shown to result primarily in apoptosis (9). Interestingly, inhibition of mono-ADP-ribosyltransferase (MART) has been shown to selectively inhibit the depolymerization of actin and formation and release of apoptotic bodies (10). Finally, most compounds that affect PARP-1 activity also affect, to various degrees, activities of other ADP-ribosyltransferases (ARTs) (11-13). With this in mind, it was the purpose of this study to establish the effect of Trp-P-1, Trp-P-2, and nine additional HCAs on the activity of PARP-1. To determine whether the effects observed were specific for PARP-1, we have tested the effects of HCAs on the arginine-specific MART A (MART-A; EC 2.4.2.31).


MATERIAL AND METHODS
The study was approved by an institutional Ethics Committee.

Enzymes and chemicals

PARP-1 was purified from bovine thymus as previously reported (14). MART-A was purified from turkey erythrocytes and donated to us by Dr. Joel Moss (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, U.S.A.) (15). [Adenosine-U-14C]NAD+ was obtained from Amersham International (Buckinghamshire, U.K.). Calf thymus DNA (type I, highly polymerized) and histone H2B were from Sigma (St. Louis, MO, U.S.A.), NAD+ from Kohjin (Tokyo, Japan), dimethyl sulfoxide (DMSO) from Nacalai Tesque (Kyoto, Japan), and HCAs were from the Japanese Cancer Association (Tokyo, Japan). All other compounds were of the best quality commercially available (usually >98% pure, according to the manufacturers' information sheets).

PARP-1 activity assay

The activity was measured as previously described (16). Briefly, PARP-1 activity was assayed by measuring the radioactivity incorporated from [adenosine-U-14C]NAD+ into trichloroacetic acid (TCA)-insoluble material. The reaction mixture (200 µl) contained 100 mM Tris/HCl (pH 8.0), 10 mM MgCl2, 5 mM dithiothreitol, 33 µg/ml DNA (sheared by sonication 10 times for 10 s), and 200 µM NAD+, including [adenosine-U-14C]NAD+. The reaction was initiated by addition of PARP-1 (0.93 µg) to the reaction mixture, carried out for 10 min at 37°C, and stopped by the addition of 0.8 ml of ice-cold 20% TCA. After standing on ice for 30 min, protein-bound [14C](ADP-ribose)n was collected on a nitrocellulose filter (Millipore; pore size, 0.45 µm) and washed 5 times with 5% TCA. After drying the filter, the acid-insoluble 14C was determined by a liquid scintillation method, in the mixture of 0.5% 2,5-diphenyloxazole and 0.03% 1,4-bis(4-methyl-5-phenyloxazol-2-yl)benzene in toluene, using a Beckman LS 5000TD counter (Beckman Instruments, Fullerton, CA, U.S.A.). The pH of the buffer was adjusted in a 1.0 M stock solution at 20°C.

MART-A activity assay

The reaction mixture for MART-A contained 50 mM potassium phosphate (pH 7.0), 100 µM [adenosine-U-14C]NAD+, 250 µg/ml histone H2B, and enzyme in 200 µl. The enzymatic reaction was carried out for 30 min at 30°C (12, 13). It was stopped and processed as described above for the PARP-1 assay. The pH of the buffer was adjusted in a 1.0 M stock solution at 20°C.

Inhibition/activation studies

The effects on PARP-1 and MART-A activities were examined under the standard conditions except for the addition of a test compound dissolved, with the exception of Trip-P-1, in DMSO. Control was the mean of duplicates with no test compound added but with an appropriate amount, if any, of DMSO. IC50 values were estimated for individual inhibitors graphically from titration curves.


RESULTS
Effects of Trp-P-1 on the activity of ARTs

Fig. 1 A shows the effects of increasing concentrations of Trp-P-1 on the activity of MART-A and PARP-1. With both enzymes, Trp-P-1 had an inhibitory effect at all concentrations tested. At the highest concentration tested (5 mM), Trp-P-1 exhibited a nearly complete inhibitory effect on PARP-1 activity [94% inhibition ()], compared to 53% with MART-A. The IC50s with Trp-P-1 were calculated at 0.22 and 2.8 mM for PARP-1 and MART-A, respectively.

Fig. 1A. Effects of Trp-P-1 and B - of Trp-P-2 on the activity of MART-A () and PARP-1 (). *Minimum value estimated under the condition of limited solubility.

Effects of Trp-P-2 on the activity of ARTs

Trp-P-2 had a mixed effect, that is, both activation and inhibition, on PARP-1, and an exclusive activating effect on MART-A at all concentrations tested (Fig. 1B). Trp-P-2 exhibited a mild inhibitory effect on PARP-1 at concentrations of 0.05 (9% ), 0.1 (12% ), and 0.5 mM (11% ). However, Trp-P-2 had an activating effect on PARP-1 at 0.75 [91% activation above controls ()], 1.0 (134% ), and 1.5 mM (70% ). At higher concentrations, Trp-P-2 only caused inhibition of PARP-1, up to 93% at 5 mM, with an IC50 of 2.2 mM. The activating effect of Trp-P-2 on MART-A was concentration-dependent up to 3 mM (>171% ) and decreased slightly at higher concentrations [e.g., 4 mM (>111% ) and 5 mM (>105% )].

Effects of various HCAs on the activity of ARTs

As a comparison to Trp-P-1 and Trp-P-2, the effects of nine other HCAs on PARP-1 and MART-A activities were determined at concentrations of 1 and 5 mM. As shown in Table 1, nine different HCAs had an inhibitory effect on PARP-1 activity at these two concentrations. The effects on MART-A (Table 2) were predominantly inhibitory with the exception of AalphaC, which slightly inhibited MART-A at 1 mM (4% ) and activated it at 5 mM (>27% ).

Table 1. Effects of various HCAs on PARP-1 activity.

a10% (final) DMSO; b2% (final) DMSO; cMinimum value estimated under the condition of limited solubility; dMaximum value estimated under the condition of limited solubility; eStimulation.

Table 2. Effects of various HCAs on MART-A activity.

a10% (final) DMSO; b2% (final) DMSO; cStimulation; dMinimum value estimated under the condition of limited solubility; eMinimum value estimated under the condition of limited solubility.

DISCUSSION
The results of the present study may shed some light on the carcinogenic potential of high doses of HCAs used in experimental systems. For instance, the carcinogenicity generally detected by long-term bioassays in rodents using doses in considerable excess of human exposure levels might be reconsidered in light of the inhibitory or stimulatory effect of HCAs on PARP-1 or MART-A. When the doses used in bioassays bring forth the coexistence of metabolized and unmetabolized HCAs, they may have a potentiating effect, that is, the metabolized HCA may cause DNA damage, while the unmetabolized HCA may inhibit PARP-1 and raise the threshold of damage required to trigger apoptosis. If this were to occur, fixation with potentially carcinogenic mutations may result in cells exposed to HCAs that would ordinarily counteract extensive DNA damage by undergoing apoptosis (17).

Alternatively, if the profound activating effect of Trp-P-2 on PARP-1, for example, occurs in vivo, an immediate decrease in available NAD+ and eventually ATP may occur, leading to an energy crisis within the cell. This may occur from Trp-P-2, the parent compound, or from DNA damage caused by its bioactivated DNA reactive metabolite. The activation of PARP-1 consumes NAD+, an essential cofactor in energy metabolism, and when PARP-1 is overactivated the depletion of both NAD+ and ATP may lead to necrotic cell death.

The cellular level of ATP is a determinant of the cell death pattern (i.e., necrosis versus apoptosis) that may result from chemical or physiological insults (18). For example, Su et al (19) have shown that PARP-1 overactivation causes extensive ADP-ribosylation of hepatocellular proteins and NAD+ depletion with the onset of necrosis in the liver of male ICR mice that obtained a high-dose of carbon tetrachloride, a non-genotoxic carcinogen. However, insults that cause a milder degree of PARP-1 activation, such as a 2-h oxygen and glucose deprivation, do not exhaust NAD+ and ATP below the level required for cells to undergo a primarily apoptotic death (20). Also, global ischemia has been shown to overactivate PARP-1 in hippocampal neurons, with the resultant onset of necrotic changes (21-23).

Finally, the biological relevance of these findings to chronic, low-level exposures to HCAs that occur in humans is uncertain. Some insight as to the relevance of previous studies showing carcinogenicity in animals treated with high-doses of HCAs was provided by Hoshi et al (24), who fed male Big Blue rats a diet with low-levels of MeIQx (named in this study 8-MeIQx) for 16 weeks. Their study showed that the no-observed-effect level (NOEL) of named in this study 8-MeIQx for in vivo mutagenicity was lower than the NOEL for in vivo carcinogenicity. The results indicate that NOELs may exist for other genotoxic carcinogens, including other types of HCAs, at low-level exposures and that adaptive or defensive processes may predominate at low-level exposures and serve to maintain biologic homeostasis. In fact, Shiotani et al (9) have shown that treatment of primary hepatocytes with Trp-P-1 at a dose as low as 30 µM causes the induction of apoptosis. Viewing the results presented herein with a threshold assumption for genotoxic carcinogens, it is possible that HCAs diminish the surveillance and strand-break detection activity of PARP-1 for basal levels of DNA damage and the damage resulting from intact or metabolically activated HCAs; the combination of which may lead to an increase in mutation frequency, possibly through cell proliferation. However, the doses at which these effects may occur in vivo and their relevance to the etiology of HCA-induced cancers are unknown.

Acknowledgements: This work was supported in part by a research fellowship from the Japan Society for the Promotion of Science (MB, RPS, and TS). The views and opinions expressed in this article are exclusively those of the authors and do not necessarily represent the views or opinions of the California Environmental Protection Agency.


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