Formation of DNA-damaging N-nitroso compounds from the interaction of calcium-channel blockers with nitrite

A large number of drugs have been shown to react with nitrite to give genotoxic-carcinogenic N-nitroso compounds (NOC). However, the majority of drugs remain to be examined in this respect, among which calcium-channel blockers, all theoretically nitrosatable and widely used in the therapy of hypertension and other cardiovascular diseases. In this preliminary investigation, seven calcium-channel blockers have been examined either for their in vitro nitrosation according to the procedure recommended by the WHO, or for occurrence of liver DNA fragmentation, as detected by the Comet assay, in rats given by gavage 1/2 LD50 of the drug and 80 mg/kg of sodium nitrite. After 6h incubation the yields of NOC formed in vitro from nicardipine, nifedipine, nimodipine and nitrendipine ranged from 37 to 45% of the theoretical one, whereas the yields of NOC formed from diltiazem, gallopamil and verapamil ranged from 2 to 5%. In vivo, as compared with the effect of the same dose of the drug alone, a significant increase of both tail length and tail moment, indicative of an increased frequency of DNA single-strand breaks and alkali-labile sites, was produced in rat liver DNA by the administration with nitrite of gallopamil, nifedipine, nimodipine and nitrendipine, the ratio [tail length of drug+NaNO(2)/tail length of drug alone] being 3.2 for nimodipine, 3.1 for gallopamil 2.2 for nifedipine, and 2.1 for nitrendipine. Even if present, the increase in the degree of DNA fragmentation did not reach the statistical significance in rats given with nitrite nicardipine, diltiazem and verapamil. Further studies should be performed to investigate the formation of NOC in conditions simulating those occurring in the stomach of humans treated with a therapeutic dose, and to quantitate their genotoxic potency.     

Chemical carcinogen in vitro testing: A method for sizing cell nuclei in the nuclear enlargement assay

The observation that cells often respond to carcinogens by nuclear enlargement has suggested that this property might be useful to develop a short-term screening test for such compounds. Previous methods for detecting nuclear size increases have used an image analyzer system to detect nuclear changes in individual cells. This paper details a more rapid method for obtaining nuclei by use of a stromalyzing procedure following by analysis of nuclear volumes, using a Coulter Counter Channelyzer. This new and simplified nuclear sizing method should facilitate the use of the assay as a possible carcinogenesis screen by permitting rapid and efficient testing of large numbers of compounds.     

Effects of tobacco smoke compounds on the ciliary activity of the embryo chicken trachea in vitro

The ciliotoxicity of 316 individual compounds representative of the gaseous and semivolatile phases of tobacco smoke has been investigated using chicken tracheal organ cultures. When examined at 5 mM concentration and measuring the time to complete ciliostasis, 36% of the compounds were found to cause ciliostasis within 15 min, while about 50% had no visible effect on the ciliary activity during a 60-min exposure. The majority of the ciliotoxic compounds were either alkylated phenylethers, benzonitriles, benzaldehydes, phenols, benzenes, naphthalenes and indoles, or α,β-unsaturated ketones and aldehydes or C₆C₁₀ aliphatic alcohols, aldehydes, acids and nitriles. Most of the compounds classified as benzoic acids, esters, polyaromatic hydrocarbons, amines and N-heterocycles, except indoles, were found to be inactive.     

Formation of mutagenic derivatives from nitrite and two primary amines

Sodium nitrite and two primary aromatic amines, viz. amino antipyrine (AAP) and aniline, were preincubated in vitro with human gastric juice. The resulting derivatives — presumably diazonium salts — were directly mutagenic in the Salmonella test. The mutagenic response was more pronounced in the case of AAP, while toxic effects narrowed the range of activity of the aniline derivative. These patterns are consistent with the findings of independent colorimetric analyses, showing that the AAP derivative is more stable at 37°C than the aniline derivative.     

Optically-based chemical and biochemical sensors for the detection of some drugs and biological compounds

The development of new methods for determining at a very low level a large spectrum of substances affecting the behaviour of living organisms is still a challenging goal. For such a purpose, chemical sensors which can be defined as the intimate combination of a sensitive and specific layer with a transducer, are undoubtedly among the more promising devices.

In this field, optical sensors are expanding rapidly, mainly based on absorption, fluorescence, chemi- and bioluminescence. Beside pH and gases, drugs (anticonvulsant, antitumour, anaesthetic …) and other compounds of biological interest can be determined with specifically designed optical sensors, for instance immunosensors.

Special attention will be given to optical biosensors with emphasis on chemi- and bioluminescence-based devices which are highly selective and ultrasensitive. When coimmobilizing various auxiliary enzymes in the sensing layer, the potentialities of such devices can be greatly extended as demonstrated by promising results recently obtained in our group.     

Effects of polychlorinated biphenyls on biological membranes: physical toxicity and molar volume relationships

Perturbation of biological membranes by a series of purified polychlorinated biphenyls (PCBs) was studied, with the objective of distinguishing nonspecific physical toxicities from specific chemical interactions. The molar volume parameter 36,000 V_x?E_B, used in the past to estimate physical toxicities and partition coefficients, was calculated for a series of PCBs. The cytotoxicity of various PCBs and other lipophilic compounds to mouse spleen lymphocytes in vitro was investigated, and ld₅₀ values were determined, using a ⁵¹Cr-release assay to measure cell viability. All PCBs tested showed similar toxicities with ld₅₀ values in the range of 2.33 × 10^?6 to 3.55 × 10^?5 M. PCBs were also capable of inhibiting the lymphocyte plasma membrane enzyme 5'-nucleotidase in vitro, with K_i values falling between 3.45 and 6.40 × 10^?5 M. When both log ld₅₀ and log K_i values were plotted against molar volume for the various compounds tested, the curves obtained followed the form predicted for a pure physical toxicity effect, with an initial linear region of slope 1 and a plateau region for compounds of large molar volume. Molar volume correlations were used to estimate the volume of lipid biophase in the systems studied and the toxic concentration of chemical in the membrane. Inhibition of plasma membrane enzymes such as 5'-nucleotidase and ATPase by PCBs is thus a nonspecific physical toxicity effect based on their lipid solubility, and there appears to be no specific chemical interaction between these molecules and membrane components.     

Comparative efficacy of novel platinum(IV) compounds with established chemotherapeutic drugs in solid tumour models

Platinum(II)-based anticancer drugs are associated with high reactivity and thus a poor biological stability. The platinum(IV)-complexes display potential advantages due to their greater stability and bioreductive activation, thereby allowing a greater proportion of the drug to arrive at the target intact. All compounds tested were able to produce cytotoxicity in monolayer cell cultures, however, the potencies of platinum(IV) drugs were lower than that observed for the platinum(II) compounds or established organic chemotherapeutic agents. There was no significant alteration in the potency of platinum(II) or (IV) compounds to produce cytotoxicity in multicellular tumour spheroids (MCTS) compared to monolayer cultures. All the organic and platinum-based cytotoxic agents produced, to varying degrees, either a retardation or reduction in MCTS growth. Proliferating cells were restricted to the outer two to three cellular layers in intermediate (d=350 microm) and large (d=600 microm) MCTS. Regardless of MCTS size, drug treatment produced a larger and more widely distributed proliferating cell population, consistent with the recruitment of quiescent cells to the proliferating pool following cytotoxic damage. Histology indicated that the predominant morphological change was that of apoptosis, although there was some drug-dependent effects such as the metaphase arrest produced by vinblastine and chromatin dispersal to the periphery of nuclei produced by doxorubicin. In summary, whilst the platinum(IV) derivatives were able to produce cytotoxicity via apoptosis, the introduction of a stable axial group significantly retarded the rate at which this occurred.     

Influence of liver S-9 preparations from rats and rainbow trout on the activity of four mutagens

The mutagenicity of four chemical compounds to strain TA100 of S. typhimurium was affected differently by liver S-9 preparations from untreated Sprague-Dawley rats and from rainbow trout (Salmo gairdneri). These two species were equally effective in decreasing the direct mutagenicity of sodium dichromate. Rat preparations were totally inactive and trout preparations were slightly active in producing mutagenic metabolites from benzo(a)pyrene (BP). Conversely, rat homogenates were significantly more efficient in activating aflatoxin B1 (AFB1) and, in particular, 2-aminofluorene (2-AF).     

Isolation and biological activity of compounds from Garcinia preussii

Context: Plants of the genus Garcinia (Clusiaceae) are traditionally used to relieve stomachaches, toothaches, and as a chew stick.

Objective: In order to determine which compounds were responsible for these activities, a phytochemical investigation of the fruits and leaves of Garcinia preussii Engl. was pursued.

Materials and methods: Plants were extracted by solvents of various polarities. Compounds isolation was then carried out using chromatography methods (medium- and high-pressure liquid chromatography, open column and thin-layer chromatography). The isolated compounds were identified and characterized by using 1D and 2D NMR spectroscopies. The antioxidant activity was evaluated using DPPH^?, ABTS^??, ALP, and ORAC assays. The antimicrobial activity was assayed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis by determining the minimum inhibitory concentration (MIC) value. The cytotoxic activity of most of the isolated compounds was evaluated on a small panel of human cancer cell lines (DU145, HeLa, HT-29, and A431) using the XTT method.

Results: The phytochemical investigation of G. preussii led to the isolation of eight known compounds, six benzophenones and two flavonoids. These compounds were tested for their biological activities. 1, 2, 3, 4, 7 and 8 demonstrated a high free radical scavenging activity with ER₅₀ ranging from 0.1 to 0.7. The antimicrobial activity was shown only against Gram-positive bacteria for 1, 4, and 5. A moderate cytotoxic activity with IC₅₀ ranging from 7 to 50?µM was observed, except for 6 which was not active.

Conclusion: These results appear to support some of the properties reported for Garcinia species.     

Methylation of liver DNA of rat and mouse by N-nitrosodimethylamine formed in vivo from dimethylamine and nitrite

The extent of formation of N-nitrosodimethylamine (NDMA) in the stomachs of rats and mice after simultaneous oral administration of [14C]dimethylamine and potassium nitrite was determined by measuring the methylation of liver DNA. With doses of around 1 mg dimethylamine hydrochloride/kg body weight and 50 mg potassium nitrite/kg body weight, 0.8% of the amine was nitrosated on average. The individual fluctuations ranged from 0.2 to 1.3% in the rat and from 0.2 to 1.9% in the mouse. Simultaneous administration of 50 mg sodium ascorbate (vitamin C)/kg body weight inhibited the nitrosation by about 80% while 50 mg alpha-tocopherol acetate (vitamin E)/kg body weight reduced the nitrosation by about a half. Assuming similar kinetics and conditions of nitrosation in rats and man, a comparison of the formation of NDMA in vivo from dietary dimethylamine and nitrite with the estimated human uptake of preformed NDMA revealed that in vivo formation in the stomach of man is probably negligible.     

Search for compounds that inhibit the genotoxic and carcinogenic effects of heterocyclic aromatic amines

Over the last 30 years approximately 160 reports have been published on dietary compounds that protect from the mutagenic and carcinogenic effects of heterocyclic aromatic amines (HAAs). In the first section of this review, the current state of knowledge is briefly summarized. Based on the evaluation of the available data, various protective mechanisms are described, and the use of different methodologies for the detection of protective effects is critically discussed. In most antimutagenicity studies (>70%) bacterial indicators (predominantly Salmonella strain TA98) were used, and about 600 individual compounds and complex mixtures have been identified that attenuate the effects of HAAs. The most frequently used in vivo method to detect protective effects are adduct measurements; anticarcinogenic dietary factors were identified by aberrant crypt foci assays and liver foci tests with rats. The mechanisms of protection include inactivation of HAAs and their metabolites by direct binding, inhibition of enzymes involved in the metabolic activation of the amines, induction of detoxifying enzymes, and interaction with DNA repair processes. The detection spectrum of conventional in vitro mutagenicity assays with metabolically incompetent indicator cells is limited. These procedures reflect only simple mechanisms such as direct binding of the HAAs to pyrroles and fibers. It has been shown that these compounds are also effective in rodents. More complex mechanisms, namely, interactions with metabolic activation reactions are not adequately represented in in vitro assays with exogenous enzyme homogenates, and false-negative as well as false-positive results may be obtained. More appropriate approaches for the detection of protective effects are recently developed test systems with metabolically competent cells such as the human Hep G2 line or primary hepatocytes. SCGE tests and DNA adduct measurements with laboratory rodents enable the detection of antigenotoxic effects in different organs, including those that are targets for tumor induction by the amines. Medium term assays based on aberrant crypt foci in colon and liver foci tests have been used to prove that certain compounds that prevented DNA damage by HAAs also reduced their carcinogenic effects. These experiments are costly and time consuming and, due to the weak induction capacity of the amines, only pronounced anticarcinogenic effects can be detected. Over the years, a large bulk of data on HAA protective compounds has accumulated, but only for a few (e.g., fibers, pyrroles, constituents of teas, and lactic acid bacteria) is there sufficient evidence to support the assumption that they are protective in humans as well.     

Studies in the metabolism of carcinogenic polycyclic heteroaromatic compounds. I. The hepatic microsomal metabolism of 7-methylbenz[c]acridine

An enzyme assay for the metabolism of the carcinogenic aza-aromatic polycyclic compound 7-methylbenz[c]acridine has been developed using a modification of a radiochemical assay described for the polycyclic aromatic hydrocarbon benzo[a]pyrene by DePierre et al. [J. W. DePierre, M. S. Moron, K. A. M. Johannesen and L. Ernster, Analyt. Biochem. 63, 470 (1975)]and Van Cantfort et al. [J. Van Cantfort, J. DeGraeve and J. E. Gielen, Biochem. biophys. Res. Commun. 79, 505 (1977)]. When the activities of control microsomes and microsomes of phenobarbital-, 3-methylcholanthrene-and 7-methylbenz[c]acridine-pretreated animals were compared, strong similarities were displayed toward oxidation of benzo[a]pyrene and 7-methylbenz[c]acridine. These similarities were seen in turnover numbers, Michaelis constants, and inducibility of both enzyme systems. 7-Methylbenz[c]acridine afforded a type I difference spectrum with 3-methylcholanthrene-pretreated microsomes. It is suggested that 7-methylbenz[c]acridine is oxidized by the same or a similar set of enzymes which is responsible for benzo[a]pyrene metabolism.     

A simplified method for the induction of 8-azaguanine resistance in Salmonella typhimurium

The selection of induced 8-azaguanine (8-AG) resistant mutants, recently developed as a test system in Salmonella typhimurium, has been adapted to the plate incorporation assay technique usually employed in the Ames test. The sensitivity of the procedure has been checked by testing known mutagenic compounds (ethyl methanesulfonate (EMS), N-methyl-N'-mtro-N-nitrosoguanidine (MNNG), 2-nitrofluorene (2-NF), 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC) and comparing the increases in mutant numbers with those obtained in the Ames test: the results so far obtained show a lower sensitivity of the 8-AG system, more notable for strains lacking R factor plasmid.Five chemicals (Natulan, Paraquat, Diquat, 4-chloro-benzotrifluoride (4-Cl-BTF), 3-nitro-4-chloro-benzotrifluoride (3-n-4-Cl-BTF), not mutagenic in the Ames test but known or suspected to be mutagenic or carcinogenic in other systems, have been also checked: two of them, the herbicides Paraquat and Diquat, were positive in the 8-AG resistance test.     

Comparison of cytotoxicity of mercury-selenium and mercury compounds on cultured cells

In vitro effects of mercury-selenium (Hg-Se) compounds, as compared with their original mercury compounds, against growth and viability of HeLa cells were studied. A water-soluble, ethanol-insoluble black complex (GX) formed from HgCl2, Na2SeO3 and GSH was less toxic than HgCl2, but the cytotoxicity of bis(methylmercuric) selenide (BMS) seemed to be similar to that of methylmercury. This may be due to the instability of BMS; i.e., there is a possibility that BMS decomposed to methylmercury and selenium under our culture conditions.