Genotoxic and genoprotective effects of 1,4-dihydropyridine derivatives: a brief review

Testing for 1,4-DHP effects in simple systems such as plasmid can reveal some interesting properties of 1,4-DHPs. Plasmid relaxation, which is indicative of DNA strand breaks, has been reported with some novel 1,4-DHP derivatives containing halogen atoms and/or nitro groups. Moreover, these compounds competed with restrictases for their binding sites (6). One of our earlier studies (13) singled out nitrendipine and AV-153-Ca and AV-153-Mg as the best DNA protectors, followed by etcarbatone and styrylcarbatone (Figure 2, previously unpublished). The effect was not concentration-dependent. In another study (15), we showed that etaftorone, fenoftorone, and cerebrocrast protected plasmid DNA against peroxynitrite-induced damage. At that point, it was believed that cerebrocrast, a lipid-soluble 1,4-DHP derivative with promising antidiabetic and neuroprotective activities but very weak Ca-channel blocking, and its analogue etaftorone act mainly on mitochondria (16–18), but both compounds turned out to be DNA binders (15).

Figure 2

The ability of 1,4-DHPs to bind to the DNA was first reported as early as in 1975 (19), suggesting an intercalative mode of DNA binding on the example of diludine and its analogues, but this research was discontinued. Diludine and its methylated analogue were shown to increase the melting temperature of DNA by 2.9 and 1.4 °C, respectively. One of our studies (20) called the attention to the DNA-binding capacity of antimutagenic AV-153-Na, as the docking experiment predicted its insertion in the DNA molecule at the place of a single-strand break, and the prediction was proven experimentally, as AV-153-Na forced ethidium bromide out of the DNA complex and showed higher affinity to damaged DNA than the intact molecule. Another, more recent study (12) has shown that DNA-binding capacity and DNA protection depend on metal ions forming salts with the AV-153 residue. In it, the fluorescence assay showed that the binding affinity dropped with each metal in the following order: Mg>Na>Ca>Li>Rb>K. Later use of spectroscopic and electrochemical methods has suggested intercalation as the main mode of action, although other types of interaction are also possible (21).

Another group of water-soluble 1,4-DHPs, i.e. carbatonides, disodium-2,6-dimethyl-1,4-dihydropyridine-3,5-bis(carbonyloxyacetate) derivatives, including carbatone, metcarbatone, etcarbatone, and styrylcarbatone have also been reported to interact with DNA but to have a lower affinity for DNA than AV-153 salts. Among them, styrylacarbatone had the highest affinity and suggested a different binding mechanism (13). This and our another study (22) also suggest that the DNA-binding affinity of water-soluble monocyclic 1,4-dihydropyridine derivatives with a carboxylate group in position 4 depends on substituents in positions 3 and 5.

We also demonstrated that 1,4-DHP derivatives, more specifically AV-153 salts, can bind to the G-quadruplexes (G4), with Na and K salts effectively binding to the human telomere repeat (18). Other authors confirmed G-quadruplex binding with a series of other novel 1,4-DHP derivatives (23).

Another recent study (24) has shown that widely used 1,4-DHPs with Ca blocker activity such as nifedipine, lercanidipine, and amlodipine can bind to the minor groove of the DNA, yet their binding affinity is low.

One of our earlier studies (16) has shown that pre-incubation with AV-153-Na (50 nmol/L) protects the DNA of HeLa cells against damage produced by peroxynitrite (200 µmol/L), as AV-153-Na penetrates the cell nucleus and up-regulates DNA excision repair enzymes. We then tested different salts of AV-153 for protectective DNA effects to learn that they were modified by the metal ions (12). AV-153-Na and AV-153-Ca effectively reduced DNA damage, AV-153-K and AV-153-Rb were less effective, whereas AV-153-Li did not protect the DNA, and AV-153-Mg even increased the damage. The AV-153 Na salt decreased DNA damage in HeLa cells through the over-expression of the pro-apoptotic protein DUX4 (Figure 3, previously unpublished). In human B-lymphocytes AV-153 Na, K, Ca, and Mg salts reduced DNA damage produced by oxidative stress caused by incorporation of the HIV-derived Tat protein (12). In another study (25), we have also found that glutapyrone protects HeLa cells against peroxynitrite, but to a lower extent than AV-153-Na.

Figure 3

Lipid-soluble cerebrocrast and etaftorone also seem to alleviate peroxynitrite-induced DNA damage (15). Cerebrocrast (50– 500 nmol/L) is effective when administered before or together with peroxynitrite, but etaftorone is effective only when given at the same time.

In a study using single-cell gel electrophoresis (the comet assay) (4), AV-153-Na was reported to reduce the number of spontaneous DNA strand breaks in HL-60 human blood lymphocytes and Raji cells. It also reduced DNA damage produced by exposure to 2 Gy of gamma-radiation, 100 µmol/L ethylmethane sulphonate (EMS), or 100 µmol/L hydrogen peroxide by up to 87 % at AV-153-Na concentrations between 1 and 10 nmol/L. A later study (3) showed that AV-153-Na stimulated the synthesis of poly(ADP-ribose) in hydrogen peroxide-treated cells and that the short-term increase in the polymer’s synthesis correlated with the higher rate and efficiency of DNA repair (3). In another study (5), AV-153-Na also reduced the number of micronucleated and apoptotic cells in a culture of human lymphocytes exposed to 2 Gy gamma radiation. The authors suggested that this effect was owed to its similar structure with the nicotinic acid and its involvement in NAD⁺ recovery and poly(ADP-ribosyl)ation (5). In a culture of Chinese hamster ovary cells (CHO-K1) treatment with AV-153-Na before exposure to X-rays did not affect the formation or repair of single-strand DNA breaks, but – judging by the FPG-modified comet assay – did prevent the formation of 8-oxoguanine (26).

When orally administered to laboratory rats (0.05 or 0.5 mg/kg) for three days, metcarbatone, etcarbatone, and styrylacarbatone caused damage to the DNA in nucleated blood cells. However, in animals with diabetes mellitus, metcarbatone and styrylcarbatone lowered DNA damage, whereas etcarbatone increased it (13). In our most recent (still unpublished) studies, oral AV-153-Na and glutapyrone also increased DNA damage in blood cells of control rats, but AV-153-Na did not significantly change the expression of the histone γ-H2AX in rat myocardium and mitigated the frequency of double-strand breaks. In another study using an experimental model of myocardial infarction (27), lacidipine decreased the level of 8-oxoguanine and therefore the DNA damage caused by oxidative stress.

Several studies using a rat streptozotocin-induced diabetes model looked into how 1,4-DHPs modify the expression of several genes and proteins involved in radical production and DNA repair (12, 15, 28). In most cases 1,4-DHPs increased the gene expression, which implies some level of influence on pathways regulating gene transcription. Other studies with diabetes mellitus have reported down-regulation of NO production and/or inducible NO synthase (iNOS) by carbatonides, etaftorone, fenoftorone, cerebrocrast (13) and AV-153 (29), which looks promising.

The first findings about the effects of 1,4-DHP derivatives on spontaneous mutations were reported for Drosophila melanogaster in 1980 by Goncharova et al. (29). The larvae fed with diludine and AV-153 had a decrease in both point mutations and chromosome aberrations. The authors concluded that these mononuclear 1,4-DHPs were more efficient antimutagens than trinuclear compounds and that their antimutagenic efficiency was related to the presence of a carboxylate group in position 4.

Studies that followed reported AV-153-Na to reduce the frequency of genetic damage (point mutations and chromosome breakage) induced by EMS in Drosophila melanogaster larvae and imagoes more efficiently than standard radioprotectors cysteine and cysteamine (30). Similar protective effect against EMS-induced mutations was reported for glutapyrone in the spermatozoa of Drosophila pre-treated at the larval stage (31). Both compounds also showed an anticlastogenic effect in in mice (32), whereas glutapyrone was also reported for an antineoplastic effect in rats exposed to continual gamma irradiation (33).

Back to Drosophila, lipid-soluble 1,4-DHPs cerebrocast and diethone (diludine) also showed antimutagenic and anticlastogenic effects (34). More recently, diludine was shown to favour gamete storage in fish (35).