Genotoxic evaluation of antiepileptic drugs by Drosophila somatic mutation and recombination test

The study examines the potential genotoxicity of three antiepileptic drugs (phenytoin sodium, pregabalin, gabapentin) using the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster. Trans-heterozygous (two genetic markers mwh and flr) third-instar larvae of D. melanogaster were treated with different concentrations of the test compounds. A positive correlation was observed between total mutations and the number of wings with morphologically detectable mutations. The observed mutations were classified according to size and type of mutation per wing. Phenytoin clearly increased the frequency of total spots at all concentrations above 1.25 μg/ml. Gabapentin also increased the frequency of total spots at concentrations of 40 and 80 μg/ml. This study shows that phenytoin and gabapentin have genotoxic effects according to the SMART test; however, pregabalin displays lower genotoxicity in the SMAR assay when compared with the other two antiepileptics. The results also show that all AED concentrations lower the survival rate of the flies.     

Genotoxic evaluation of sodium fluoride in the Somatic Mutation and Recombination Test (SMART)

In this study, genotoxic effect of sodium fluoride (NaF) was investigated in Drosophila melanogaster Somatic Mutation and Recombination Test. Third-instar larvae trans-heterozygous for two genetic markers mwh and flr, were treated at different concentrations (2.5 μg/ml, 5 μg/ml and 10 μg/ml) of the test compounds. After the treatment the observed mutations were classified according to size and type of mutation per wing. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which is distilled water. NaF has genotoxic and toxic effects for concentrations of 5 and 10 μg/ml. The present study shows that NaF may have genotoxic and toxic effects.     

Genotoxicity studies in the ST cross of the Drosophila wing spot test of sunflower and soybean oils before and after frying and boiling procedures

Sunflower and soybean oils were tested for genotoxicity in the Drosophila wing somatic mutation and recombination assay. Results indicate that both oils produce genotoxic effects when tested without any previous frying or boiling processes. Boiling sunflower oil during fifteen, thirty and sixty minutes significantly increased its genotoxic response; nevertheless, after frying potatoes this oil showed a significant decrease in the genotoxic activity. On the other hand, boiling and frying soybean oil in the same conditions results in a decrease of its genotoxic potential. We have also detected that the amount of total polar materials increases significantly in oils submitted to frying or boiling process. Nevertheless, in oils obtained after frying potatoes, the amount of TPM was higher than after boiling. It is suggested that this effect is probably due to the amount of non-volatile TPM, the fatty acid composition of the oils, the types of frying oil, the high frying temperature and time, and the number of boiling and frying. This is the first study reporting genotoxicity data in Drosophila for the boiling and frying of both sunflower and soybean oils.     

Genotoxicity studies of organically grown broccoli (Brassica oleracea var. italica) and its interactions with urethane,methyl methanesulfonate and 4-nitroquinoline-1-oxide genotoxicity in the wing spot test of Drosophila melanogaster

Broccoli (Brassica oleracea var. italica) has been defined as a cancer preventive food. Nevertheless, broccoli contains potentially genotoxic compounds as well. We performed the wing spot test of Drosophila melanogaster in treatments with organically grown broccoli (OGB) and co-treatments with the promutagen urethane (URE), the direct alkylating agent methyl methanesulfonate (MMS) and the carcinogen 4-nitroquinoline-1-oxide (4-NQO) in the standard (ST) and high bioactivation (HB) crosses with inducible and high levels of cytochrome P450s (CYPs), respectively. Larvae of both crosses were chronically fed with OGB or fresh market broccoli (FMB) as a non-organically grown control, added with solvents or mutagens solutions. In both crosses, the OGB added with Tween–ethanol yielded the expected reduction in the genotoxicity spontaneous rate. OGB co-treatments did not affect the URE effect, MMS showed synergy and 4-NQO damage was modulated in both crosses. In contrast, FMB controls produced damage increase; co-treatments modulated URE genotoxicity, diminished MMS damage, and did not change the 4-NQO damage. The high dietary consumption of both types of broccoli and its protective effects in D. melanogaster are discussed.     

Influence of CYP2B6 and ABCB1 SNPs on nevirapine plasma concentrations in Burundese HIV-positive patients using dried sample spot devices

AIMS

The pharmacokinetics (PK) and pharmacogenetics (PG) of nevirapine have been studied in rich and limited-resource countries. CYP2B6 single nucleotide polymorphisms (SNPs) have been associated with decreased drug clearance. We evaluated the PG determinants of nevirapine trough concentrations in a rural cohort in Burundi using easy to store and transport dried sample spot devices.

METHODS

A cross-sectional analysis in HIV-positive nevirapine-treated patients in Kiremba, north of Burundi, was performed in 2009. After blood withdrawal whole blood was stored on dried blood spots and plasma (after centrifugation) was placed on dried plasma spot devices and stored at room temperature. Nevirapine plasma and dried sample spot concentrations were compared to test the clinical usefulness of this method. SNPs in CYP2B6 and ABCB1 (using a real time PCR technique) were analyzed and associated with nevirapine plasma trough concentrations.

RESULTS

Nevirapine concentrations measured on dried plasma spot devices were highly related to plasma concentrations in 60 patients, although a negative bias was observed (−18%). Nevirapine trough concentrations were above the target concentration (3000 ng ml⁻¹) in 84% of patients and they were associated with CYP2B6 SNPs (both at position 516 and 983). No effect of ABCB1 SNPs was noted.

CONCLUSIONS

Dried plasma spot devices are accurate tools for measuring nevirapine concentrations in rural settings where refrigeration is not available, despite a moderate underestimation bias. They allowed the evaluation of nevirapine concentrations in a cohort of HIV-infected people in rural Burundi, confirming very good exposure and correlation with PG polymorphisms in the CYP2B6 encoding gene.     

Associations of ABCB1, NFKB1, CYP3A,and NR1I2 polymorphisms with cyclosporine trough concentrations in Chinese renal transplant recipients

Aim:

Cyclosporine requires close therapeutic drug monitoring because of its narrow therapeutic index and marked inter-individual pharmacokinetic variation. In this study, we investigated the associations of CYP3A4, CYP3A5, ABCB1, NFKB1, and NR1I2 polymorphisms with cyclosporine concentrations in Chinese renal transplant recipients in the early period after renal transplantation.

Methods:

A total of 101 renal transplant recipients receiving cyclosporine were genotyped for CYP3A4^*1G, CYP3A5^*3, ABCB1 C1236T, G2677T/A, C3435T, NFKB1 −94 ins/del ATTG, and NR1I2 polymorphisms. Cyclosporine whole blood levels were measured by a fluorescence polarization immunoassay. Trough concentrations of cyclosporine were determined for days 7-18 following transplantation.

Results:

The dose-adjusted trough concentration (C₀) of cyclosporine in ABCB1 2677 TT carriers was significantly higher than that in GG carriers together with GT carriers [90.4±24.5 vs 67.8±26.8 (ng/mL)/(mg/kg), P=0.001]. ABCB1 3435 TT carriers had a significantly higher dose-adjusted C₀ of cyclosporine than CC carriers together with CT carriers [92.0±24.0 vs 68.4±26.5 (ng/mL)/(mg/kg), P=0.002]. Carriers of the ABCB1 1236TT-2677TT-3435TT haplotype had a considerably higher CsA C₀/D than carriers of other genotypes [97.2±21.8 vs 68.7±26.9 (ng/mL)/(mg/kg), P=0.001]. Among non-carriers of the ABCB1 2677 TT and 3435 TT genotypes, patients with the NFKB1 −94 ATTG ins/ins genotype had a significantly higher dose-adjusted C₀ than those with the −94 ATTG del/del genotype [75.9±32.9 vs 55.1±15.1 (ng/mL)/(mg/kg), P=0.026].

Conclusion:

These results illustrate that the ABCB1 and NFKB1 genotypes are closely correlated with cyclosporine trough concentrations, suggesting that these SNPs are useful for determining the appropriate dose of cyclosporine.     

Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS

Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of ³⁴S labeled dimethylthioarsinic acid (³⁴S-DMTA^V) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched ³⁴S-DMTA^V made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS^V). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, ³⁴S-DMTA^V underwent several transformations. Labile ³⁴S was exchanged with more abundant ³²S to produce ³²S-DMTA^V, a thiol group was added to yield DMDTA^V, and a methyl group was added to yield ³⁴S-TMAS^V. Because incubation of ³⁴S-DMTA^V resulted in the formation of ³⁴S-TMAS^V, the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS^V from dimethylarsinic acid (DMA^V) would proceed via a dimethylarsinous acid (DMA^III) intermediate and would necessitate the loss of ³⁴S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA^V to TMAS^V. Additionally, the detection of isotopically pure ³⁴S-TMAS^V raises questions about the sulfur exchange properties of TMAS^V in the cecum material. Therefore, ³⁴S-TMAS^V was incubated and the exchange was monitored with respect to time. The data suggest that the As-S bond associated with TMAS^V is less labile than the As-S bond associated with DMTA^V.     

Chronic arsenic exposure increases TGFalpha concentration in bladder urothelial cells of Mexican populations environmentally exposed to inorganic arsenic

Inorganic arsenic (iAs) is a well-established carcinogen and human exposure has been associated with a variety of cancers including those of skin, lung, and bladder. High expression of transforming growth factor alpha (TGF-alpha) has associated with local relapses in early stages of urinary bladder cancer. iAs exposures are at least in part determined by the rate of formation and composition of iAs metabolites (MAs(III), MAs(V), DMAs(III), DMAs(V)). This study examines the relationship between TGF-alpha concentration in exfoliated bladder urothelial cells (BUC) separated from urine and urinary arsenic species in 72 resident women (18-51 years old) from areas exposed to different concentrations of iAs in drinking water (2-378 ppb) in central Mexico. Urinary arsenic species, including trivalent methylated metabolites were measured by hydride generation atomic absorption spectrometry method. The concentration of TGF-alpha in BUC was measured using an ELISA assay. Results show a statistically significant positive correlation between TGF-alpha concentration in BUC and each of the six arsenic species present in urine. The multivariate linear regression analyses show that the increment of TGF-alpha levels in BUC was importantly associated with the presence of arsenic species after adjusting by age, and presence of urinary infection. People from areas with high arsenic exposure had a significantly higher TGF-alpha concentration in BUC than people from areas of low arsenic exposure (128.8 vs. 64.4 pg/mg protein; p<0.05). Notably, exfoliated cells isolated from individuals with skin lesions contained significantly greater amount of TGF-alpha than cells from individuals without skin lesions: 157.7 vs. 64.9 pg/mg protein (p=0.003). These results suggest that TGF-alpha in exfoliated BUC may serve as a susceptibility marker of adverse health effects on epithelial tissue in arsenic-endemic areas.     

Effects of Li(+) transport and Li(+) immobilization on Li(+)/Mg(2+) competition in cells: implications for bipolar disorder

Li(+)/Mg(2+) competition has been implicated in the therapeutic action of Li(+) treatment in bipolar illness. We hypothesized that this competition depended on cell-specific properties. To test this hypothesis, we determined the degree of Li(+) transport, immobilization, and Li(+)/Mg(2+) competition in lymphoblastomas, neuroblastomas, and erythrocytes. During a 50 mM/L Li(+)-loading incubation, Li(+) accumulation at 30 min (mmoles Li(+)/L cells) was the greatest in lymphoblastomas (11.1+/-0.3), followed by neuroblastomas (9.3+/-0.5), and then erythrocytes (4.0+/-0.5). Li(+) binding affinities to the plasma membrane in all three cell types were of the same order of magnitude; however, Li(+) immobilization in intact cells was greatest in neuroblastomas and least in erythrocytes. When cells were loaded for 30 min in a 50 mM/L Li(+)-containing medium, the percentage increase in free intracellular [Mg(2+)] in neuroblastoma and lymphoblastoma cells ( approximately 55 and approximately 52%, respectively) was similar, but erythrocytes did not exhibit any substantial increase ( approximately 6%). With the intracellular [Li(+)] at 15 mM/L, the free intracellular [Mg(2+)] increased by the greatest amount in neuroblastomas ( approximately 158%), followed by lymphoblastomas ( approximately 75%), and then erythrocytes ( approximately 50%). We conclude that Li(+) immobilization and transport are related to free intracellular [Mg(2+)] and to the extent of Li(+)/Mg(2+) competition in a cell-specific manner.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

Inhibitory effects of water extract of propolis on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster.

Propolis is a substance produced by honeybees (Apis mellifera L.). Its components are strong antioxidants and free radical scavengers. The aim of this study was to evaluate the protective effects of a water extract of Brazilian green propolis (WEP) combined with the antitumor agent doxorubicin (DXR) on Drosophila melanogaster wing cells through the somatic mutation and recombination test (SMART). Two different crosses were used: The standard (ST) cross and the high bioactivation (HB) cross. The HB cross is characterized by a constitutively enhanced level of cytochrome P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Larvae obtained from these two crosses were chronically treated with different concentrations of WEP (12.5,25.0 and 50.0 mg/mL) alone or combined with DXR (0.125 mg/mL). The results obtained with the two different crosses were rather similar. Neither toxicity nor genotoxicity were observed in WEP treated series. Simultaneous treatment with different concentrations of WEP and DXR led to a reduction in the frequency of recombination compared to the treatment with DXR alone. This anti-recombinogenic effect was proportional to the concentrations applied, indicating a dose-response correlation and can be attributed to the powerful scavenger ability of WEP.     

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 microM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 microM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca(2+) and non-Ca(2+) divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 microM MeHg, 97.7% of Purkinje cells were viable. At 3 microM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 microM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca(2+) divalent cation released by MeHg in Purkinje neurons.     

Effect of arvanil (N-arachidonoyl-vanillyl-amine), a nonpungent anandamide-capsaicin hybrid,on ion currents in NG108-15 neuronal cells

The effects of arvanil (N-arachidonoyl-vanillyl-amine), a structural hybrid between capsaicin and anandamide, on ion currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. Arvanil (0.2-50 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Arvanil produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of arvanil-induced inhibition of I(Ca,L) was 2 microM. Arvanil (5 microM) could shift the steady-state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. No effect of arvanil (20 microM) on delayed rectifier K(+) current (I(K(DR))) was observed; however, capsaicin (20 microM), glyceryl nonivamide (20 microM) and capsinolol (20 microM) suppressed it significantly. Arvanil (20 microM) caused a slight reduction in the amplitude of erg (ether-à-go-go-related)-mediated K(+) current (I(K(erg))) without modifying the activation curve of this current, while capsaicin and glyceryl nonivamide were more effective in suppressing I(K(erg)). Under current-clamp configuration, arvanil decreased the firing frequency of action potentials. Arvanil-mediated inhibition of I(Ca,L) appeared to be independent of its binding to either vanilloid or cannabinoid receptors. The channel-blocking properties of arvanil may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.     

Ecotoxicological assessment of bromobenzene using a test battery with five model systems.

Bromobenzene (BrB) is used as a solvent for crystallization and as an additive to motor oils and may be released into the environment through various waste streams. However, there is limited available information about the toxic hazard of BrB in the aquatic environment. Consequently, the ecotoxicological effects induced by BrB were investigated using five model systems with representants from four trophic levels. The battery included bioluminescence inhibition of the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris and immobilization of the cladoceran Daphnia magna. Total protein content, neutral red uptake and MTS metabolization were reduced, while lysosomal function, succinate dehydrogenase activity, G6PDH activity and leakage, metallothionein levels and EROD activity were stimulated in PLHC-1 and RTG-2 fish cell lines. The most sensitive bioindicator was the bioluminiscence of V. fischeri, with an EC(50) of 0.04mM BrB at 15min and a non-observed adverse effect level of 0.02 mM BrB. There is a large difference in sensitivity to BrB among the model systems probably due to the metabolic capacity of the different species. PLHC-1 cells were more sensitive to BrB than RTG-2 cells. The most prominent morphological effects observed were hydropic degeneration, loss of cells and of the perinuclear pattern of distribution of lysosomes. Therefore, BrB should be classified as toxic to aquatic organisms.     

Effect of gender and cigarette smoking on urinary excretion of etheno DNA adducts in humans measured by isotope dilution gas chromatography/mass spectrometry

Endogenous formation of the promutagenic DNA adducts 1,N(6)-ethenoadenine (epsilon Ade) and 3,N(4)-ethenocytosine (epsilon Cyt) has been considered as biomarkers originated from lipid peroxidation. Elevated levels of epsilon Ade and epsilon Cyt were observed in cancer-prone tissues, suggesting the validity of these adducts in cancer risk assessment. The presence of DNA base adducts in biological fluids is considered to derive primarily from base excision repair (BER) systems. In this study, a modified gas chromatography/mass spectrometry (GC/MS) method is developed for simultaneous analysis of epsilon Ade and epsilon Cyt in human urine. After adjusting for creatinine concentration, urinary excretion of epsilon Ade, as well as epsilon Cyt, is much higher in 18 male smokers than in 10 male nonsmokers (p=0.003 for epsilon Ade and p=0.04 for epsilon Cyt). Furthermore, excretion of epsilon Ade and epsilon Cyt in 14 female nonsmokers is much higher than in 10 male nonsmokers (p=0.002 for epsilon Ade and p=0.005 for epsilon Cyt). These results suggest a statistically significant association between gender, as well as smoking, and excretion of epsilon Ade and epsilon Cyt. Moreover, urinary excretion of epsilon Ade in these 42 subjects correlates with that of epsilon Cyt (R(2)=0.6846, p<0.0001). Measurement of urinary epsilon Ade and epsilon Cyt excretion should provide valid noninvasive biomarkers for carcinogenesis and chemoprevention studies.     

Chlorpyrifos and its metabolites alter gene expression at non-cytotoxic concentrations in D3 mouse embryonic stem cells under in vitro differentiation: Considerations for embryotoxic risk assessment

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10 μM CPF and TClP and 100 μM CPO for 12 h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20 μM CPF and 25 μM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.     

Interactions of sulforaphane and dimethyl sulfoxide with methyl methanesulfonate,urethane, 4-nitroquinoline-1-oxide and hydrogen peroxide in the Drosophila melanogaster wing spot test

Sulforaphane (SF) is an isothiocyanate present in Brassicaceae, vegetables that induce the detoxification of electrophiles and reactive oxygen species. SF has been correlated with chemoprevention mechanisms against degenerative diseases. We tested if the SF had an effect against methyl methanesulfonate (MMS), urethane (URE), 4-NQO and H₂O₂. SF (>95% purity, 0.14, 0.28, 0.56 mM) was diluted in a DMSO/Tw80/EtOH mixture (DTE) corresponding to 25, 50, 100% of lyophilized broccoli. The SF treatment (0.14 mM) was positive for small spots in the ST cross and negative in the HB cross. In the HB cross, SF (0.28 mM) was genotoxic. In the ST cross, the SF treatments showed a tendency to reduce the genotoxic damage caused by MMS, which could be explained by the radical scavenging action of the DTE mixture. In the ST cross, the frequency of small spots in the SF 0.14 mM/URE treatment was similar to that of Water/URE, which can be explained by a DTE and SF scavenger action. In both crosses, the results for the direct oxidants, 4-NQO and H₂O₂, were different and must be related to differential modulation of CYPs expression and the SF and DTE scavenger properties.     

S0859, an N-cyanosulphonamide inhibitor of sodium-bicarbonate cotransport in the heart

BACKGROUND AND PURPOSE: Intracellular pH (pH(i)) in heart is regulated by sarcolemmal H(+)-equivalent transporters such as Na(+)-H(+) exchange (NHE) and Na(+)-HCO(3) (-) cotransport (NBC). Inhibition of NBC influences pH(i) and can be cardioprotective in animal models of post-ischaemic reperfusion. Apart from a rabbit polyclonal NBC-antibody, a selective NBC inhibitor compound has not been studied. Compound S0859 (C(29)H(24)ClN(3)O(3)S) is a putative NBC inhibitor. Here, we provide the drug's chemical structure, test its potency and selectivity in ventricular cells and assess its suitability for experiments on cardiac contraction. EXPERIMENTAL APPROACH: pH(i) recovery from intracellular acidosis was monitored using pH-epifluorescence (SNARF-fluorophore) in guinea pig, rat and rabbit isolated ventricular myocytes. Electrically evoked cell shortening (contraction) was measured optically. With CO(2)/HCO(3) (-)-buffered superfusates containing 30 muM cariporide (to inhibit NHE), pH(i) recovery is mediated by NBC. KEY RESULTS: S0859, an N-cyanosulphonamide compound, reversibly inhibited NBC-mediated pH(i) recovery (K (i)=1.7 microM, full inhibition at approximately 30 microM). In HEPES-buffered superfusates, NHE-mediated pH(i) recovery was unaffected by 30 microM S0859. With CO(2)/HCO(3) (-) buffer, pH(i) recovery from intracellular alkalosis (mediated by Cl(-)/HCO(3) (-) and Cl(-)/OH(-) exchange) was also unaffected. Selective NBC-inhibition was not due to action on carbonic anhydrase (CA) enzymes, as 100 microM acetazolamide (a membrane-permeant CA-inhibitor) had no significant effect on NBC activity. pH(i) recovery from acidosis was associated with increased contractile-amplitude. The time course of recovery of pH(i) and contraction was slowed by S0859, confirming that NBC is a significant controller of contractility during acidosis. CONCLUSIONS AND IMPLICATIONS: Compound S0859 is a selective, high-affinity generic NBC inhibitor, potentially important for probing the transporter's functional role in heart and other tissues.