Factors influencing the microsome- and mitochondria-catalyzed in vitro binding of diethylnitrosamine and N-nitrosopiperidine to deoxyribonucleic acid.

[¹⁴C]Diethylnitrosamine ([¹⁴C]DEN) and [¹⁴C]N-nitrosopiperidine ([¹⁴]NPiP) bind covalently to calf thymus DNA in an in vitro incubation system containing rat liver microsomes. The reaction is NADPH-dependent. Pretreatment of the animals with phenobarbital (PB) enchances the binding of both DEN and NPiP to DNA, whereas the binding of DEN to DNA decreases after 3-methylcholanthrene pretreatment. The PB effect, as observed from the binding of DEN to DNA. is more pronounced in young rats than in the older animals. Addition of cytosol to the incubation system enhances the binding of DEN 3- to 4-fold and the binding of NPiP 2- to 3-fold. Addition of mitochondria to the incubation system increases the binding of [¹⁴C]DEN only slighty. but increases the binding of NPiP more than 5-fold. Addition of mitochondria has no effect on the binding of [¹⁴C]dimethylnitrosamine ([¹⁴C]DMN). Mitochondria alone markedly catalyze the binding of NPiP to DNA. Addition of benzylamine. which is a substrate of mitochondrial monoamine oxidase as well as an inhibitor of DMN-demethylase, inhibits the binding of NPiP catalyzed by microsomes and microsomes plus mitochondria.     

The hepatocarcinogenicity of diethylnitrosamine to rainbow trout and its enhancement by Aroclors 1242 and 1254

Rainbow trout (Salmo gairdneri) were fed diets containing 100 ppm Aroclor 1242 (AC42) or Aroclor 1254 (AC54) in combination with 1100 ppm diethylnitrosamine (DEN) for one year. The incidence of hepatocarcinomas was determined and compared with the incidence in trout fed 1100 ppm DEN alone. The two Aroclors dramatically enhanced tumor incidence from 10.2% in the positive controls (DEN alone) to 40.2% for AC42 and 21.6% for AC54. This is in contrast to previous results obtained when AC54 was fed concomitantly with aflatoxin B1 (AFB1), where a substantial inhibition of carcinogenesis was observed. The alteration of chemical carcinogenesis in trout by PCB, therefore, depends upon the carcinogen involved and is not a generalized effect.     

Immunohistochemical detection of melphalan-DNA adducts in colon cancer cells in vitro and human colorectal liver tumours in vivo

Melphalan is a chemotherapeutic drug that exerts its cytotoxic effect mainly through the formation of DNA adducts. We report the specific immunohistochemical detection and visualisation of melphalan-DNA adducts using the monoclonal antibody MP5/73 in cultured tumour cells and solid tumour tissue from colorectal liver metastases from patients treated with melphalan. The human colon cancer cell lines HT29, SW480 and SW1116, and the rat colon cancer cell line CC531 were exposed to different concentrations of melphalan. In addition, tumour samples from 17 patients with colorectal liver metastases treated by isolated hepatic perfusion with high dose melphalan (200mg) were collected. Cell lines and tumour samples were stained with the MP5/73 antibody against melphalan-DNA adducts and cell viability was determined by an MTT assay. Melphalan-DNA adducts could be visualised by immunohistochemistry in both cultured cells and solid tumour tissue. A correlation between melphalan exposure concentration, the subsequent melphalan-DNA adduct staining intensity, and melphalan cytotoxicity existed for each individual cell line, but the level of both parameters independently differed between cell lines. Specific staining for melphalan-DNA adducts also was feasible in the human solid tumour tissue. There was considerable variation in melphalan-DNA adduct staining, staining intensity, and distribution in the tumour stroma and the tumour epithelium among the different patients. Melphalan-DNA adducts appeared to be more intense in tumour cells at the border of the tumour nodules than in tumour cells in the centre. Thus, visualisation of melphalan-DNA adducts by immunohistochemistry allows the study of distribution of melphalan-DNA adducts in solid tumours.     

Genotoxic thresholds,DNA repair,and susceptibility in human populations

It has been long assumed that DNA damage is induced in a linear manner with respect to the dose of a direct acting genotoxin. Thus, it is implied that direct acting genotoxic agents induce DNA damage at even the lowest of concentrations and that no “safe” dose range exists. The linear (non-threshold) paradigm has led to the one-hit model being developed. This “one hit” scenario can be interpreted such that a single DNA damaging event in a cell has the capability to induce a single point mutation in that cell which could (if positioned in a key growth controlling gene) lead to increased proliferation, leading ultimately to the formation of a tumour.     

Effects of dimethylnitrosamine on some actions of testosterone

Using homogenates of mouse kidney and testes, the activities of the enzymes, β-glucuronidase and β-galactosidase, were studied as markers of androgen action. The results obtained differed between testes and kidney homogenates. Dimethylnitrosamine (DMN) may cause a competetitive inhibition of the anabolic action of testosterone in kidney homogenates but this was not evident from the results obtained with testes homogenates.     

Lead acetate does not inhibit dimethylnitrosamine activation and interacts with phenobarbital which is genotoxic in the ST cross of the Drosophila wing spot test

Lead acetate (PbAc) is known to inhibit the synthesis of the heme group, needed for hemeproteins like Cytochromes P450 (CYP450s). Dimethylnitrosamine (DMN) requires metabolic activation by CYP450s. The Drosophila wing spot test was performed to establish whether PbAc inhibits DMN activation in the standard (ST) and high bioactivation (HB) crosses, with different levels of CYP450s. Phenobarbital (PH) was used as an antagonist for its ability to induce CYP450s synthesis. PbAc (0.01, 0.1, 1.0 mM) produced significant small spots frequencies in the ST cross, indicating a possible genotoxic activity, however, the total spots frequency was negative at all concentrations. DMN (0.076 mM) was genotoxic in both crosses; surprisingly, PH (12 mM) was genotoxic and the PH-DMN treatment resulted synergic in the ST cross. Interestingly, the PbAc-PH pre-co-treatments showed a possible interaction in the ST cross. The GC-MS analysis showed a drop in the PH content as the PbAc concentration increased. PbAc also seemed to inhibit the genotoxic activity of PH, except at 0.01 mM. It is concluded that PbAc does not inhibit DMN activation by CYP450s in both crosses since it exerted a clear genotoxicity and that PH is genotoxic and interacts with PbAc in the ST but not the HB cross.     

Possible prevention from the progression of cardiotoxicity in adriamycin-treated rabbits by coenzyme Q10

The cumulative dose-dependent cardiotoxicity induced by doxorubicin (adriamycin, ADR) and its possible prevention by coenzyme Q₁₀ (CoQ₁₀) were studied in rabbits. In the group that received ADR alone, ADR dose-dependent electrocardiography (ECG) abnormalities and severe myocardial damage on electron microscopic examination were observed. In the group that received ADR + CoQ₁₀, these alterations occurred in lesser degree, and ECG changes seemed to be improved. The results indicated that CoQ₁₀ might prevent the progression of cardiotoxicity in ADR-treated rabbits.     

Role of zinc and iron chelation in apoptosis mediated by tachpyridine, an anti-cancer iron chelator

Tachpyridine (N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane; tachpyr) is a potent hexadentate iron chelator under preclinical investigation as a potential anti-cancer agent. Tachpyridine induces apoptosis in cultured cancer cells by triggering a mitochondrial pathway of cell death that is p53-independent. To explore the relationship between the chelation chemistry of tachpyridine and its biological activity, a sensitive and specific reversed-phase high-performance liquid chromatography (RP-HPLC) method was devised and used to measure tachpyr and its metal complexes in cells and tissue culture media. Major species identified in cells treated with tachpyr were tachpyr itself, [Zn(tachpyr)](2+), and iron coordinated to two partially oxidized species of tachpyridine, [Fe(tachpyr-ox-2)](2+), and [Fe(tachpyr-ox-4)](2+). The kinetics of intracellular accumulation of [Zn(tachpyr)](2+) and [Fe(tachpyr-ox-2)](2+) were markedly different: [Zn(tachpyr)](2+) rapidly reached plateau levels, whereas intracellular levels of [Fe(tachpyr-ox-2)](2+) and free tachpyr rose steadily. At the last timepoint measured, 9% of total cellular iron and 13% of total cellular zinc were bound by tachpyridine. Taken together, [Zn(tachpyr)](2+), [Fe(tachpyr-ox-2)](2+), and free tachpyr accounted for virtually all of the tachpyr added, indicating that iron and zinc are the principal metals targeted by tachpyridine in cells. Consistent with these findings, activation of the apoptotic caspases 9 and 3 was blocked in cells pre-treated with either iron or zinc. Pretreatment with either of these metals also completely protected cells from the cytotoxic effects of tachpyridine. These results demonstrate a link between metal depletion and chelator cytotoxicity, and suggest that intracellular chelation of zinc as well as iron may play a role in the cytotoxicity of tachpyridine.     

Reversible inhibition of DNA, RNA and protein synthesis in human cells by lead chloride.

An acute intoxication by lead chloride (conc. 2.5 - 10(-4)M) produces a temporary reduction of macromolecular syntheses in HeLa cells growing asynchronously. The reduction is similar for DNA, RNA and proteins and differs only in the intensity. After a one-day intoxication, if the cells are put back in a fresh medium, the syntheses return to normal within 10 h. The histochemical sulphide-silver method shows that lead is present in the cells during the inhibition.     

Cytotoxicities of the l and d isomers of phenylalanine mustard in L1210 cells

The cytotoxicity of d-phenylalanine mustard (medphalan) to murine L1210 leukemia cells in culture was reduced by both the d and l isomers of leucine. l-Leucine only partially protected L1210 cells from medphalan cytotoxicity at drug concentration's above 10 μM, suggesting that medphalan uptake occured via both an amino acid carrier and an as yet undetermined agency, possibly passive diffusion. At equitoxic concentrations of l-phenylalanine mustard (melphalan) and medphalan, l-leucine reduced medphalan cytotoxicity by only one-sixth that obtained with melphalan. Analysis of melphalan and medphalan inhibition of the initial rate of l-leucine transport indicated a melphalan K_i of 0.085 mM, a value one-seventh that of melphalan (K_i, 0.635 mM).     

Hemolytic activity of aqueous extract of Livistona chinensis fruits.

Livistona chinensis is used as an anticancer agent in traditional Chinese medicine. In vitro, the extracts of fruits and seeds of L. chinensis are known to possess antiangiogenic and antiproliferative activities. Here we report the presence of phenolic compounds in L. chinensis fruits which show hemolytic activity. The hemolytic activity of phenolics is limited to an acid-precipitable fraction. Further, presence of proteins and lipids abrogated the hemolytic activity indicating astringent and membrane damaging activities as mechanisms of hemolysis. In conclusion, the hemolytic activity of phenolics in L. chinensis fruits is due to astringent and membrane damaging activities.     

A short-term test to detect organotropic specificity of carcinogens

Transplacental administration of 200 mg/kg of 3,4-benzpyrene (Bp) by intraperitoneal injection caused marked increase in 8-azaguanine (8AG)-resistant mutations in lung (75.210⁷ cells) and total body cells. Morphological transformations and chromosome aberrations were also observed in lung and total body cells. On transplacental administration of 200 200 mg/kg of dimethylnitrosamine (DMN), the highest rates of morphological transformation (6.53%) and 8AG-resistant mutation (12810⁷ cells) were induced in cells of liver origin, and chromosome abnormalities were also detected in liver and total body cells. Transplacental administration of 100 mg/kg of nitrosomethylurea (NMU) caused a marked increase in transformed colonies (4.08–4.40%) and 8AG-resistant mutations (134.8 and 79.910⁷ cells) of brain and total body cells. The cells from mice and rats that were most affected by transplacental administration of these chemicals were derived from the respective target organs of these chemicals in vivo.     

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.     

Effects of capsaicin on P-gp function and expression in Caco-2 cells

Capsaicin is the pungent component of hot chilli, a popular spice in many populations. The aim of the present study was to evaluate the chronicity and reversibility of the modulating effect of capsaicin on both the P-gp expression and activity in the Caco-2 cell monolayers. Capsaicin at concentrations ranging from 10 to 100 microM, which were found to be non-cytotoxic towards the Caco-2 cells, were observed to inhibit P-gp mediated efflux transport of [3H]-digoxin in the cells. The acute inhibitory effect was dependent on the capsaicin concentration and duration of exposure, with abolishment of polarity of [3H]-digoxin transport attained at 50 microM of capsaicin. In contrast, longer term (48 and 72 h) co-incubation of the Caco-2 cells with capsaicin (50 and 100 microM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by higher degree of [3H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. The induction of P-gp protein and mRNA levels was also influenced by capsaicin concentration and duration of exposure, with higher expression levels, in particular of the mRNA, seen at higher spice concentrations over prolonged period of incubation. Our data suggest that caution should be exercised when capsaicin is to be consumed with drugs that are P-gp substrates. In particular, the oral bioavailability of these drugs may be influenced by the P-gp status of populations that rely heavily on hot chilli in their diets.     

Melatonin-mediated regulation of human MT(1) melatonin receptors expressed in mammalian cells

In mammals, the pineal hormone melatonin activates G protein-coupled MT(1) and MT(2) melatonin receptors. Acute exposure of recombinant MT(1) and MT(2) melatonin receptors to supraphysiological concentrations of melatonin differentially regulates these two receptors with the MT(2), but not the MT(1), exhibiting rapid desensitization and internalization. In the present study, we sought to determine whether prolonged exposure to supraphysiological and physiological concentrations of melatonin desensitized and/or internalized the MT(1) melatonin receptor. Using a Chinese hamster ovary (CHO) cell line stably expressing MT(1)-FLAG or transiently expressing MT(1)-green fluorescent protein (GFP) melatonin receptors, we found that prolonged exposure (8h) to supraphysiological concentrations of melatonin (100 nM) significantly increased the number of MT(1) melatonin receptors and decreased the affinity (K(i)) of melatonin for competition for 2-[125]iodomelatonin. A similar treatment also desensitized the MT(1) melatonin receptor-mediated stimulation of [(35)S]GTPgammaS binding, but did not internalize the receptor. In contrast, prolonged exposure to a concentration of melatonin mimicking nocturnal levels (400 pM) did not affect the number of MT(1) melatonin receptors, the affinity for melatonin, or the functional sensitivity of the receptor. We conclude that in vivo endogenous melatonin does not significantly affect the functional sensitivity of MT(1) melatonin receptors, however, exogenous melatonin taken therapeutically at doses above physiological levels could desensitize the receptor thereby affecting physiological responses mediated following activation of MT(1) melatonin receptors.     

Kinetics of micronucleus induction and cytotoxicity caused by distinct antineoplastics and alkylating agents in vivo

This mini-review aims to compare the differences in the kinetics of the induction of micronucleated polychromatic erythrocytes (MN-PCE) and cytotoxicity by distinct antineoplastic and genotoxic agents in murine peripheral blood in vivo and to correlate these kinetics with the underlying processes. Comparisons were carried out using our previously obtained data with nominal doses causing similar levels of cytotoxicity, as measured in terms reduction of PCE. The aneuploidogens caused the most rapid induction of MN-PCEs and had the highest rates of cytotoxicity and genotoxicity. The promutagens cyclophosphamide and dimethylnitrosamine showed the most delayed responses and had the lowest genotoxic and cytotoxic efficiencies. DNA crosslinking agents had a similar delay of 4–5 h, greater than those of aneuploidogens, but differed in their cytotoxic and genotoxic efficiencies. Methylnitrosourea and 5-aza-cytidine caused greater delays than crosslinking agents. These delays can be due to the methylnitrosourea-mediated induction of formation of mono alkyl adducts which are interpreted as mismatches during DNA duplication, whereas 5-aza-cytidine requires incorporation into the DNA to induce breakage. This review allows us to conclude that the requirement for metabolic activation and the mechanisms of DNA breakage and of micronucleus induction are the main factors that affect the time of maximal MN-PCE induction.     

Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats

Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) was given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and β-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P < 0.001) reduced the number and size of nodules formed. Also, a significant (P < 0.01) reduction in serum activities of AST, ALT, ALP, LDH and γGT was noticed. Expression of COX-2 and NFkB p65 was significantly reduced whereas that of p53, Bax and caspase 3 increased at the mRNA and protein levels. Likewise, a decrease in levels of β-arrestin and the anti-apoptotic marker Bcl-xL was also noted. These findings suggest that chrysin exerts global hepato-protective effect and its chemopreventive activity is associated with p53-mediated apoptosis during early hepatocarcinogenesis.     

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

Cytotoxicity and genotoxicity of nitrogen dioxide (NO₂) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO₂ in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract.Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO₂, 0.1 ppm NO₂, 1 ppm NO₂, 10 ppm NO₂ and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively.The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO₂ in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO₂ in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.     

Influence of royal jelly on the reproductive function of puberty male rats

The adverse effects of royal jelly on the reproductive system of puberty male rats were investigated. Royal jelly was daily administered by gavage to Sprague–Dawley rats at doses 200, 400, and 800 mg/kg for 4 weeks. The body weight and organ coefficients were determined. Sperm count, spermatozoa abnormality, and testicular histopathology were examined through light microscopy. Radioimmunoassay was used to detect serum hormones. The dietary exposure to royal jelly did not affect body weight, but the organ coefficients for the pituitary and testis in the high-dose group were decreased significantly compared with the control group, and significant changes in the microstructure of the testis were observed. No significant differences in sperm count were observed among all groups, however, the sperm deformity rate in the high-dose group increased significantly. Serum hormones in the high-dose group were significantly different from the control group. After royal jelly was stopped for 14 days, the adverse changes were partially reversed and returned to levels close to those in the control group. In conclusion, high-dose royal jelly oral administration for 4 weeks adversely affected the reproductive system of pubescent male rats, but the unfavorable effects are alleviated to some extent by cessation of administration.     

Enhanced chemiluminescence of phagocytic cells from rats treated with chlorphentermine

The chronic administration of chlorphentermine to rats resulted in the appearance of enlarged, foamy alveolar macrophages (AMs) m the alveoli of the lungs. Circulating polymorphonuclear leukocytes (PMNs) from treated rats did not show a change in size. When challenged with unopsonized zymosan, the chemiluminescence (CL) of AMs from treated rats was enhanced with the peak response occurring later than with control AMs. Opsonization of the zymosan led to a further increase in CL from the foamy AMs while having no effect with control AMs. PMNs from treated rats also gave an enhanced CL response when compared to PMNs from control rats.