Induction of gene mutations and gene conversions by vinyl chloride metabolites in yeast.

Chloroethylene oxide and 2-chloroacetaldehyde, two metabolites of vinyl chloride, and 2-chloroethanol, a putative metabolic intermediate, were assayed for their genetic activity in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Chloroethylene oxide was found to be the most effective in inducing forward mutations in Sch. pombe and gene conversions in S. cerevisiae, increasing the mutation and conversion frequencies 340 and 50 times, respectively, over those of the controls. In either the presence or the absence of mouse liver microsomes, 2-chloroacetaldehyde showed only feeble genetic activity, and 2-chloroethanol was completely inactive in both yeast strains. In contrast to vinyl chloride, 2-chloroacetaldehyde did not induce forward mutations in Sch. pombe inthe host-mediated assay in mice. The results strongly support the hypothesis that chloroethylene oxide is one of the principal mutagenic agents formed from vinyl chloride in the presence of mouse liver enzymes.

Favorable effects of flecainide in transvenous internal cardioversion of atrial fibrillation.

OBJECTIVES: The aim of the study was to evaluate the effects of intravenous (IV) flecainide on defibrillation energy requirements in patients treated with low-energy internal atrial cardioversion. BACKGROUND: Internal cardioversion of atrial fibrillation is becoming a more widely accepted therapy for acute episode termination and for implantable atrial defibrillators. METHODS: Twenty-four patients with atrial fibrillation (19 persistent, 5 paroxysmal) underwent elective transvenous cardioversion according to a step-up protocol. After successful conversion in a drug-free state, atrial fibrillation was induced by atrial pacing; IV flecainide (2 mg/kg) was administered and a second threshold was determined. In patients in whom cardioversion in a drug-free state failed notwithstanding a 400- to 550-V shock, a threshold determination was attempted after flecainide. RESULTS: Chronic persistent atrial fibrillation was converted in 13/19 (68%) patients at baseline and in 16/19 (84%) patients after flecainide. Paroxysmal atrial fibrillation was successfully cardioverted in all the patients. A favorable effect of flecainide was observed either in chronic persistent atrial fibrillation (13 patients) or in paroxysmal atrial fibrillation (5 patients) with significant reductions in energy requirements for effective defibrillation (persistent atrial fibrillation: 4.42+/-1.37 to 3.50+/-1.51 J, p < 0.005; paroxysmal atrial fibrillation: 1.68+/-0.29 to 0.84+/-0.26 J, p < 0.01). In 14 patients not requiring sedation, the favorable effects of flecainide on defibrillation threshold resulted in a significant reduction in the scores of shock-induced discomfort (3.71+/-0.83 vs. 4.29+/-0.61, p < 0.005). No ventricular proarrhythmia was observed for any shock. CONCLUSIONS: Intravenous flecainide reduces atrial defibrillation threshold in patients treated with low-energy internal atrial cardioversion. This reduction in threshold results in lower shock-induced discomfort. Additionally, flecainide may increase the procedure success rate in patients with chronic persistent atrial fibrillation.

CYP superfamily perturbation by diflubenzuron or acephate in different tissues of CD1 mice.

This work aimed to investigate whether the insecticide acephate (125 or 250 mg/kg b.w.) or diflubenzuron (752 or 1075 mg/kg b.w.), two of the most widely used pesticides worldwide, impairs CYP-linked murine metabolism in liver, kidney and lung microsomes after repeated (daily, for three consecutive days) i.p. administration. The regio- and stereo-selective hydroxylation of testosterone was used as multibiomarker of different CYP isoforms. Both gender and tissue specific effects were observed. Lung was the most responsive tissue to induction by lower diflubenzuron dose, as exemplified by the marked increase of testosterone 7alpha-hydroxylation (CYP2A) (up to 13-fold) in males. Higher dose produced a generalized inactivation. At the lower dose acephate induced 6beta- (CYP3A1/2, liver) as well as 2beta- (CYP2B1/2, kidney) hydroxylase activities ( approximately 5 and approximately 4-fold increase, respectively) in males. In females, a marked suppression of the various hydroxylations was observed. At 250 mg/kg of acephate, animals did not survive. Induction of the most affected isoforms was sustained by immunoblotting analysis. Corresponding human CYP modulations might disrupt normal physiological functions related to these enzymes. Furthermore, the co-mutagenic and promoting potential of these pesticides, phenomena linked to CYP upregulation (e.g. increased bioactivation of ubiquitous pollutants and generation of oxygen free radicals) are of concern for a more complete definition of their overall toxicological potential.

Third International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis.

This conference, attended by scientists from 27 countries, focused on the most recent advances in the field of antimutagenesis and anticarcinogenesis. Particular emphasis was given to the mechanistic approach, which is believed to be an essential prerequisite for a safer and more effective implementation of chemoprevention of cancer and of mutation-related diseases. The arrangement of the six regular sessions basically followed and updated the detailed classification of mechanisms of inhibitors of mutagenesis and carcinogenesis proposed by S. De Flora and C. Ramel (Mutat. Res., 202: 285-306, 1988), covering both extracellular and cellular mechanisms involved in the prevention of mutations and cancer initiation, as well as in the modulation of later stages of the carcinogenesis process. In addition, a workshop was devoted to methodological aspects concerning the modulation of the genotoxic and carcinogenic response. The present report covers the main themes of overview lectures or research communications presented by more than 60 speakers. Most presentations were multi-authored, as the result of collaborative studies, in several cases at the international level, but only the names of speakers will be given.

Efficacy and tolerability in fully conscious patients of transvenous low-energy internal atrial cardioversion for atrial fibrillation.

Transvenous low-energy atrial cardioversion was performed in a series of fully conscious patients (30 patients with chronic atrial fibrillation and 5 patients with paroxysmal atrial fibrillation). The results show that internal atrial defibrillation is effective and tolerable in most patients.

Left superior vena cava persistence in patients undergoing pacemaker or cardioverter-defibrillator implantation: a 10-year experience.

OBJECTIVE: The persistence of a left superior vena cava (LSVC) has been observed in 0.3% of the general population as established by autopsy. In the adult population, it is an important anatomic finding if a left superior approach to the heart is considered. The aim of the study was to evaluate the prevalence of a LSVC in patients undergoing pacemaker (PM) and cardioverter-defibrillator (CD) implantation. DESIGN: We observed the prevalence of LSVC during a 10-year period; each patient undergoing PM or transvenous CD implantation received a left cephalic/left subclavian venous approach to the heart. With this technique, LSVC persistence is easily diagnosed during lead placement. RESULTS: A total of 1,139 patients consecutively underwent PM implantation during 10 years: 4 patients had persistent LSCV (0.34%). Among 115 patients undergoing CD implantation, 2 patients with LSVC (1.7%) were observed. Overall LSVC persistence was found in 6 of 1,254 patients (0.47%). Two patients, one of whom had no right superior vena cava (RSVC), received a left-sided PM, whereas two other patients received right-sided devices. Both CD patients received a left-sided active-can device: the first patient with a right-sided lead tunneled to the left pectoral pocket, as a result of poor catheter handling through the LSVC and coronary sinus, and the second patient with a screw-in lead from LSVC. Long-term follow-up of these patients (average +/- SD, 41 +/- 26 months) revealed absence of lead dislodgment and appropriate device function regardless of lead implantation site. CONCLUSIONS: Persistence of LSVC in adults undergoing PM/CD implantation is similar to that of the general population (0.47% in our study). The left-sided implant can be achieved by stylet shaping and by use of active fixation leads in most patients, with a reliable outcome at short term in addition to appropriate device performance at follow-up. Assessment of the RSVC is advisable when planning a right-sided implantation, since a minority of patients lacks this vessel.

Evaluation of myocardial injury following repeated internal atrial shocks by monitoring serum cardiac troponin I levels.

INTRODUCTION: Electrical shocks delivered for atrial cardioversion (CV) may cause myocardial damage. The aim of this study was to assess the extent of myocardial injury caused by repeated intracardiac shocks delivered for low-energy internal atrial CV. METHODS AND RESULTS: Thirty-five patients with chronic persistent atrial fibrillation (AF) of different etiologies underwent CV with delivery of synchronized biphasic shocks (3.0/3.0 ms) between two catheters positioned in the right atrium and the coronary sinus. Shocks were delivered according to a step-up protocol (50 V, 180 V, then steps of 40 to 56 V up to 500 V, if necessary). In 23 patients, AF was reinduced after baseline CV, and CV was repeated. Myocardial injury was monitored by measuring cardiac troponin I (cTnI) serum concentrations in blood samples taken at baseline and at 2, 4, 8, 12, and 24 h after the procedure, by means of an immunoenzymologic assay (normal values, < or =0.6 ng/mL). A mean (+/- SD) of 6.9 +/- 3.4 shocks per patient were delivered (range, 2 to 17). Shocks delivered in each patient had a maximal energy of 7.3 +/- 4.0 J (range, 1.7 to 15.7). In 20 patients (57%), no evidence of myocardial injury (cTnI level, < or = 0.6 ng/mL) was found. In 13 patients (37%), mildly elevated cTnI levels (range, 0.7 to 1.4 ng/mL) in samples taken 4 to 12 h after CV suggested minor myocardial injury. In two patients (6%), higher cTnI levels were found in samples taken 4 to 8 h after CV (peak, 1.7 and 2.4 ng/mL), indicating a necrotic damage. Patients with no cTnI elevation, with mild cTnI elevation, or with cTnI levels >or =1.5 ng/mL did not differ significantly with respect to the total number of shocks delivered, the mean amount of energy delivered, and the cumulative amount of energy delivered. No clinical complications were observed. CONCLUSIONS: Following internal CV with the delivery of repeated shocks, minor elevations of cTnI serum levels could be detected in a significant proportion of patients, and this suggests subtle asymptomatic minor myocardial injury. The elevations of cTnI levels do not appear to be related to the number of shocks or to the amount of energy delivered.

Effects of spermine on formation of HGPRT- mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C in V79 Chinese hamster cells.

Spermine is a polyamine found in bacteria, animal, and plant tissues. It is involved in a variety of biological processes, and its interaction with DNA stabilizes the secondary structure of the double helix. Spermine is one of the first reported antimutagens, reducing the mutation rate in several prokaryotic test systems, while in eukaryotic organisms conflicting results have been obtained. In light of the significant antimutagenic effect of spermine, it is important to evaluate its activity in mammalian cells in culture. The present study was undertaken to evaluate the ability of spermine to suppress the level of HGPRT- mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C. Spermine reduced the mutation frequency induced by ethylmethanesulfonate and methylmethanesulfonate but did not affect survival; with mitomycin C survival was reduced but mutation rate was not influenced.

Malignant vasovagal syncope: a randomised trial of metoprolol and clonidine.

OBJECTIVE: To evaluate the efficacy of head up tilt guided treatment with metoprolol and clonidine in preventing the recurrence of syncope in patients with malignant vasovagal syncope. PATIENTS: 20 patients (9 men and 11 women, mean age 33 (SD 17), range 14 to 62 years) with severe symptoms. DESIGN: Randomised double blind crossover trial; efficacy was assessed by head up tilt testing. RESULTS: Metoprolol was more effective than clonidine in abolishing syncope (19/20 v 1/20, P < 0.001) but clonidine showed some beneficial effects on time to syncope and severity of hypotension in 12 patients. During an average follow up of 15 (3) months there was a significant reduction in the recurrence of symptoms compared with the previous year in patients who had tilt up guided treatment (18 metoprolol, 1 clonidine). CONCLUSIONS: Treatment guided by head up tilting is a reliable method of treating patients with malignant vasovagal syndrome. Metoprolol was an effective long term treatment for preventing syncope. High doses were more effective and a careful dose titration period helped to minimise withdrawal symptoms and side effects.

Electrophysiological effects of flecainide and propafenone on atrial fibrillation cycle and relation with arrhythmia termination.

OBJECTIVES: (1) To investigate the electrophysiological effects of flecainide and propafenone during atrial fibrillation, and their relation to arrhythmia termination; (2) to investigate the effects of isoprenaline on atrial fibrillation in basal conditions and during treatment with class 1C drugs to evaluate the role of adrenergic stimulation on proarrhythmic events occurring during this treatment. DESIGN: Prospective, single centre study. SETTING: University hospital. METHODS: 10 patients with lone paroxysmal atrial fibrillation underwent an electrophysiological study. The dynamic behaviour of MFF (the mean of 100 consecutive atrial fibrillation intervals) was evaluated at two atrial sites after induction of atrial fibrillation either at baseline or after class 1C drug administration (flecainide or propafenone 2 mg/kg). The effects of isoprenaline on MFF and RR interval were also investigated both under basal conditions and during class 1C drug treatment. RESULTS: After induction of atrial fibrillation, mean (SD) MFF shortened with time, and was further shortened by isoprenaline infusion (177 (22) v 162 (16) v 144 (11) ms, p < 0.05). The administration of class 1C drugs reversed this trend and significantly increased the MFF to an average of 295 (49) ms, leading to conversion to sinus rhythm within 10 minutes in all patients. Atrial fibrillation was then reinduced on class 1C drugs: isoprenaline shortened the MFF and RR interval with a trend to AV synchronisation (223 (43) v 269 (49) ms for the MFF, 347 (55) v 509 (92) ms for the RR, p < 0.05); 1:1 sustained AV conduction occurred in two patients, at 187 and 222 beats/min respectively. One of these patients underwent electrical cardioversion because of haemodynamic collapse. CONCLUSIONS: Class 1C drugs are effective at restoring sinus rhythm by increasing the MFF to a much greater extent than observed in self terminating atrial fibrillation episodes, and reversing the spontaneous atrial fibrillation behaviour (progressive shortening of MFF and self perpetuation of atrial fibrillation). MFF prolongation with 1:1 conduction at fast ventricular rates may lead to synchronisation during adrenergic stimulation, with a very short ventricular cycle; hence it is advisable to keep the patients at rest after acute class 1C drug loading or to consider pharmacological modulation of AV conduction for patients who are prone to a fast ventricular response.

Metabolism of 5-methoxypsoralen by Saccharomyces cerevisiae.

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.

Effects of cinnamaldehyde on survival and formation of HGPRT- mutants in V79 cells treated with methyl methanesulfonate, N-nitroso-N-methylurea, ethyl methanesulfonate and UV light.

The effects of the antimutagenic flavoring cinnamaldehyde on the induction of HGPRT- mutants by methyl methanesulfonate (MMS), N-nitroso-N-methylurea (MNU), ethyl methanesulfonate (EMS) and UV light was investigated in the Chinese hamster V79 cell line. Cinnamaldehyde did not show any mutagenic or toxic effects in this experimental system by itself and did not modify mutation frequency when given to cells simultaneously with chemical mutagens. Under these conditions, the cytotoxicity of MMS, but not that of MNU and EMS, was increased. Cell viability was also reduced in MNU-, EMS-, or UV light-pretreated cells when they were postincubated in the presence of cinnamaldehyde. Moreover, cinnamaldehyde reduced the frequency of mutations induced by MMS but not by the other mutagens. The results obtained suggest that cinnamaldehyde inhibits some cellular function(s) promoting the repair of a variety of different cytotoxic lesions. At the same time, stimulation by cinnamaldehyde of an error-free DNA repair mechanism acting on MMS-induced mutagenic damage was indicated.

Genetic toxicity of procarbazine in bacteria and yeast.

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride] is used to treat Hodgkin's disease. This compound was tested in vitro without and with S10 fraction from mice liver (microsomal assay) using Saccharomyces cerevisiae strain D7, Salmonella typhimurium (strains TA98, TA100, TA1535) and in vivo in Swiss albino mice (host-mediated assay) using D7. Procarbazine, without S10 fraction, is highly toxic and induced mitotic crossover, gene conversion, and reverse mutation in D7. It had a toxic effect on all the Salmonella strains; but did not induce reverse mutations at the histidine loci. Procarbazine, with S10 fraction, was less toxic and did not induce genetic effects in yeast or Salmonella. In the host-mediated assay, no genetic effects were seen.

Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS.

At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol. It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency. This increase is not observed for point mutation. By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM). This treatment does not modify the frequencies of point mutation. These data support the notion that an inducible recombinogenic function exists in wild-type yeast.

Antimutagenicity in yeast.

In recent years there has been increasing interest in antimutagenesis, and studies have been done using both prokaryotic and eukaryotic systems. In eukaryotic systems the first studies were performed with different strains of Schizosaccharomyces pombe. In particular, caffeine and L-methionine were investigated. Different strains of Saccharomyces cerevisiae were employed in studies of a wide variety of compounds, including acridine, saccharin, salts, tumor promoters and co-carcinogens. Strain D7 was widely employed and antimutagenic activity of spermine, chlorophyllin, cobaltous chloride and fermented milk is reported.

Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae.

Potassium dichromate and chromium chloride were analyzed for their ability to induce mitotic gene conversion and point reverse mutation in D7 diploid strain of S. cerevisiae. We used cells from the stationary phase of growth with and without metabolic activation (S9 hepatic fraction) and cells from the logarithmic phase, that contain a high level of cytochrome P-450 and have a greater permeability. In the present work we confirmed the genetic activity of K2Cr2O7 in cells from the stationary phase, with and without S9 fraction and in cells from the logarithmic growth phase. A slight increase in genetic activity was observed in experiments with CrCl3 using phosphate buffer, but no genetic effects were noted in Tris-HCl buffer. Our studies suggest that phosphate ion may be the carrier responsible of the entrance of trivalent chromium in the cells. The higher cellular permeability may account for the different results obtained with both compounds in cells from the stationary and logarithmic phases of growth.

Inhibition of yeast cytochrome P-450 by ammonium metavanadate.

Incubation of diploid D7 strain cells of Saccharomyces cerevisiae (grown in 20% glucose) in the presence of ammonium metavanadate (AMV) led to a decrease in the cytochrome P-450-dependent monooxygenase system (cytochrome P-450 level and 7-ethoxycoumarin O-deethylase). The electrophoretic analysis of microsomal fractions of yeast cells treated with metavanadate revealed a decrease in the intensity of the bands corresponding to a M(r) in the range of 51,000-58,000 Da compared with those observed in controls, i.e., cells grown in 20% glucose. Analysis of the cytochrome P-450 transcript showed that AMV treatment reduced the mRNA level. Our results suggest that AMV inhibits the yeast cytochrome P-450 system by acting at both the pre- and post-transcriptional levels.

Genetic activity of 2-aminofluorene in the salmonella/erythrocyte mutagenicity assay.

In a previous study, we demonstrated the activation of cyclophosphamide by mouse erythrocytes in a yeast test using the D7 strain of Saccharomyces cerevisiae. The present study provides further information on the ability of washed red blood cells from mice to activate 2-aminofluorene (2-AF) detected as an increase in mutation frequency of the tester strain, TA1538 (frameshift mutation) of Salmonella typhimurium. The 2-AF was tested at different concentrations (1-8 micrograms/plate) using both the liquid-suspension test and the agar-plate test. For comparison, the bioactivation of 2-AF by the hepatic postmitochondrial supernatant (S9 fraction) from Aroclor-1254-induced rats was studied. 2-AF was only found to be clearly mutagenic in the agar-plate test with both activation systems. The genetic response obtained with the erythrocytes appeared to be related to the number of cells/plate. At the lowest dose, slight differences are observed when genotoxic effects were compared to those with the S9 fraction.

Influence of cinnamaldehyde on UV-induced gene conversion and point mutation in yeast: effect on protein synthesis.

The activity of cinnamaldehyde (CIN), a bioantimutagen in bacterial systems, was tested in the D7 strain of yeast Saccharomyces cerevisiae. Yeast cells were UV-irradiated and post-incubated in liquid growth medium for 2 and 4 h with different concentrations of cinnamaldehyde. During the post-incubation period, DNA-damage-specific functions may be induced. This in turn may affect the genotoxicity and in fact a weak decrease in UV-induced convertant and revertant frequencies was observed after 4 h of post-incubation. The presence of CIN in the growth medium increased the UV-induced gene conversion and reversion. The addition of cycloheximide abolished this effect. To evaluate the CIN effect on protein synthesis, extracts of cells UV-treated and post-incubated for 2 h in the presence of 35S-methionine were performed. SDS-gel electrophoresis demonstrated the inhibitory effect of CIN on a UV-specific protein. This work suggests that CIN might interfere with DNA-damage-inducible systems although it did not exert an antimutagenic activity in our experimental conditions.

EDTA affects cytochrome P450-dependent biotransformation reactions during incubations for the liver microsomal assay.

In order to optimize the condition of the liver microsomal assay (LMA), studies were carried out to determine the effects of EDTA on mixed-function oxidase activity and its stability under the exact incubation conditions for the LMA. Aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from beta-naphthoflavone and sodium phenobarbital (beta-NF + PB)- or Aroclor 1254 (AC)-treated mice were examined during a period of preincubation with EDTA ranging from 1 to 40 mM. At 5 mM EDTA, we obtained a strong inhibition of the microsomal LP as well as the greatest value of the mean specific activity (Asp) for both APD and pNAD activities. In agreement with the biochemical data, the presence of 5 mM EDTA in the incubation mixtures for the LMA significantly increased the mitotic gene conversion, mitotic crossing-over and point-reverse mutation of the well-known premutagen cyclophosphamide (30 mM) on the diploid D7 strain of Saccharomyces cerevisiae as the outcome of a greater metabolic activity. We concluded that the systematic use of 5 mM EDTA in LMA mixtures could improve the reliability and sensitivity of such a test.