Disulfiram-based disulfides as narrow-spectrum antibacterial agents

Sixteen disulfides derived from disulfiram (Antabuse?) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic parameters over disulfiram.     

Disulfiram inhibition of the alternative respiratory pathway in plant mitochondria

Disulfiram (tetraethylthiuram disulfide) was found to be a potent and selective inhibitor of the alternative respiratory path of plant mitochondria. The onset of inhibition by disulfiram takes several minutes and the inhibition is not readily reversed by washing, nor by metal ions. By contrast, thiols such as dithiothreitol not only reverse, but also prevent, disulfiram inhibition. Inhibition by disulfiram and by hydroxamic acids are not mutually exclusive. Structural analogs of disulfiram are far less potent inhibitors, with the exception of bisethyl xanthogen. Inhibition is due to disulfiram, per se, and not to its reduction product, diethyldithiocarbamate, a powerful chelator. Accordingly, the inhibitory effect of disulfiram is considered to involve the formation of mixed disulfides with one or more sulfhydryl groups in the alternative path. Disulfiram does not act as an electron sink diverting electron flow from oxygen.     

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram

The NAD⁺-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD⁺ protected almost 100% at 10 μM but only 50% at 5 mM, and vice versa if the enzyme was pre-incubated with NAD⁺ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.     

The inhibition of lipid peroxidation by disulfiram prevents the killing of cultured hepatocytes by allyl alcohol, tert-butyl hydroperoxide, hydrogen peroxide and diethyl maleate

Disulfiram is a potent antioxidant that prevented the peroxidation of microsomal phospholipids induced by ADP/Fe3+ at concentrations as low as 1 microM. However, disulfiram had a biphasic action when used to assess the role of lipid peroxidation in the killing of cultured hepatocytes by an acute oxidative stress. At a relatively low concentration (10 microM), the antioxidant activity of disulfiram predominated, and there was protection against the killing of the hepatocytes by allyl alcohol, tert-butyl hydroperoxide, hydrogen peroxide, and diethyl maleate. As the concentration of disulfiram was increased above 10 microM, the extent of protection progressively decreased. Thus, with higher concentrations of disulfiram, there was a second action whose consequence is to obscure the protective effect of the lower doses. With the agents studied, this additional and as yet undefined action of disulfiram leads to the killing of the hepatocytes by a mechanism that is unrelated to the peroxidation of lipids. This biphasic action of disulfiram must be appreciated in any attempt to use this compound to assess the role of lipid peroxidation in toxic cell injury.     

Disulfiram is a potent inhibitor of rat 5-lipoxygenase activity

The effect of disulfiram on the 5-lipoxygenase activity from rat polymorphonuclear leukocyte cell-free lysates was determined and compared with that of other thiocarbamoyl and aryl disulfides. Disulfiram was a potent inhibitor of the soluble 5-lipoxygenase causing 50% inhibition at submicromolar concentrations (0.4-0.7 microM). The inhibition by disulfiram was similar to that of bis(diisopropylthiocarbamoyl) disulfide with both compounds being about 100-fold more potent as inhibitors than the structurally related bis(4-methyl-1-homopiperazinylthiocarbonyl) disulfide analog. The potency of 5-lipoxygenase inhibition by disulfiram was comparable to that of diphenyldisulfide (IC50 = 0.2-0.4 microM), in the same range or better than most typically used inhibitors. However, the degree of inhibition by disulfiram was more sensitive to thiols than that of diphenyldisulfide, as shown by the selective protection against disulfiram inhibition by low concentrations of thiols. Diethyldithiocarbamate, the reduction product of disulfiram, was a less potent inhibitor of the 5-lipoxygenase activity, causing only a partial inhibition (40-60%) over a wide range of concentrations (2-30 microM). The results demonstrate that disulfiram is a potent inhibitor of 5-lipoxygenase in vitro and provide the basis for further investigations on the effect of the drug on leukotriene biosynthesis inhibition and its contribution to the ethanol-disulfiram reaction. They also indicate that disulfiram represents a sensitive reagent to characterize the thiol requirement of the 5-lipoxygenase reaction.     

Chronic Inhibition of Dopamine β-Hydroxylase Facilitates Behavioral Responses to Cocaine in Mice

The anti-alcoholism medication, disulfiram (Antabuse), decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using DBH knockout (Dbh −/−) mice, disulfiram, and the selective DBH inhibitor, nepicastat. Locomotor activity was measured in control (Dbh +/−) and Dbh −/− mice during a 5 day regimen of saline+saline, disulfiram+saline, nepicastat+saline, saline+cocaine, disulfiram+cocaine, or nepicastat+cocaine. After a 10 day withdrawal period, all groups were administered cocaine, and locomotor activity and stereotypy were measured. Drug-naïve Dbh −/− mice were hypersensitive to cocaine-induced locomotion and resembled cocaine-sensitized Dbh +/− mice. Chronic disulfiram administration facilitated cocaine-induced locomotion in some mice and induced stereotypy in others during the development of sensitization, while cocaine-induced stereotypy was evident in all nepicastat-treated mice. Cocaine-induced stereotypy was profoundly increased in the disulfiram+cocaine, nepicastat+cocaine, and nepicastat+saline groups upon cocaine challenge after withdrawal in Dbh +/− mice. Disulfiram or nepicastat treatment had no effect on behavioral responses to cocaine in Dbh −/− mice. These results demonstrate that chronic DBH inhibition facilitates behavioral responses to cocaine, although different methods of inhibition (genetic vs. non-selective inhibitor vs. selective inhibitor) enhance qualitatively different cocaine-induced behaviors.     

Chemical Biology Drug Sensitivity Screen Identifies Sunitinib as Synergistic Agent with Disulfiram in Prostate Cancer Cells

Background

Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects.

Methods and Findings

In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression.

Conclusions

Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell growth. Disulfiram-sunitinib combination was identified as one of the potent synergistic approaches. Since sunitinib alone has been reported to lack efficacy in prostate cancer clinical trials, our results provide a rationale for novel combinatorial approach to target prostate cancer more efficiently.     

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.     

Acidification and sodium entry in frog skin epithelium

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na⁺ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na⁺ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na⁺ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO₂; and (d) the positive correlation between net Na⁺ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.     

Inhibition of phytoalexin synthesis in arachidonic Acid-stressed potato tissue by inhibitors of lipoxygenase and cyanide-resistant respiration

Arachidonic acid-stressed potato tuber discs synthesized the phytoalexin rishitin. This synthesis was inhibited by salicylhydroxamic acid (SHAM), and to a lesser extent by tetraethylthiuram disulfide (disulfiram). Disulfiram was less effective apparently because it was inactivated in the tuber discs. Disulfiram and SHAM both inhibited cyanide-resistant respiration of whole potato discs and lipoxygenase extracted from these discs. When low disulfiram concentrations were used, the lipoxygenase inhibition was quickly overcome, again because the disulfiram apparently was inactivated by oxidation.     

Leaf litter decomposition as a bioassessment tool of acidification effects in streams: Evidence from a field study and meta-analysis

Atmospheric acid deposition affects many streams worldwide, leading to decreases in pH and in base cations concentrations and increases in aluminum (Al) concentration. These changes in water chemistry induce profound changes in the diversity, structure and activity of biological communities and in ecosystem processes. However, monitoring programs rely only on chemical and structural indicators to assess stream integrity. Nevertheless, the ability of ecosystems to provide services rely on their functional integrity and thus ecosystem processes should be considered in monitoring programs. We assessed the potential for leaf litter decomposition, a fundamental ecosystem process in forest streams, to be used as a bioassessment tool of acidification effects on stream ecosystem functioning. In a field study in the Vosges Mountains (North-eastern France), using three leaf litter species (Alnus glutinosa, Acer pesudoplatanus and Fagus sylvatica) enclosed in fine and coarse mesh bags and incubated in streams flowing over granite or sandstone bedrock along an acidification gradient, we assessed if the response of litter decomposition to acidification depended on litter species, mesh size, parent lithology and acidification level. In a meta-analysis of 17 primary studies on the effect of acidification on leaf litter decomposition, reporting 67 acidified – reference stream comparisons, we assessed the consistency in the response of litter decomposition to acidification cross studies and the robustness of litter decomposition to be used as a bioassessment tool. Both the field study and meta-analysis revealed an overall strong inhibition (>60%) of leaf litter decomposition in acidified streams likely resulting from previously well described altered decomposer community structure and activity. No effect of leaf species was found in the field study, while in the meta-analysis inhibition of leaf litter decomposition in acidified streams was stronger for Fagus than for Acer, Quercus and Liriodendron. However, differences among leaf species in the meta-analysis might have been confounded by other differences among studies. The response of leaf litter decomposition to acidification was stronger in coarse than in fine mesh bags, indicating strong impairment of detritivore community structure and activity. The magnitude of inhibition also depended on parent lithology, but this is likely related to differences in the degree of acidification. Indeed, the magnitude of the inhibition of leaf litter decomposition increases with increases in H⁺ in Al concentration. Litter decomposition has the potential to be used as a bioassessment tool of acidification effects in streams since it shows consistent response to acidification across regions and is robust to experimental choices.     

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC₅₀ values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.     

Impaired Leaf Litter Processing in Acidified Streams

Anthropogenic acidification in headwater streams is known to affect microbial assemblages involved in leaf litter breakdown. Far less is known about its potential effects on microbial enzyme activities. To assess the effects of acidification on microbial activities associated with decaying leaves, a 70-day litter bag experiment was conducted in headwater streams at six sites across an acidification gradient. The results revealed that microbial leaf decomposition was strongly and negatively correlated with total Al concentrations (r?=??0.99, p?<?0.001) and positively correlated with Ca²⁺ concentrations (r?=?0.94, p?=?0.005) and pH (r?=?0.93, p?=?0.008). Denaturing gradient gel electrophoresis analyses showed that microbial assemblages differed between non-impacted and impacted sites, whereas fungal biomass associated with decaying leaves was unaffected. The nutrient content of leaf detritus and ecoenzymatic activities of carbon (C), nitrogen (N) and phosphorus (P) acquisition revealed that N acquisition was unaltered, while P acquisition was significantly reduced across the acidification gradient. The P content of leaf litter was negatively correlated with total Al concentrations (r?=??0.94, p?<?0.01) and positively correlated with decomposition rates (r?=?0.95, p?<?0.01). This potential P limitation of microbial decomposers in impacted sites was confirmed by the particularly high turnover activity for phosphatase and imbalanced ratios between the ecoenzymatic activities of C and P acquisition. The toxic form of Al has well-known direct effects on aquatic biota under acidic conditions, but in this study, Al was found to also potentially affect microbially mediated leaf processing by interfering with the P cycle. These effects may in turn have repercussions on higher trophic levels and whole ecosystem functioning.     

Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria

• 1.1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro.
• 2.2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate.
• 3.3. Mitochondrial respiration inhibition was dependent upon alteration of —SH groups.
• 4.4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane.
• 5.5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

     

Response of Copepods to Elevated pCO2 and Environmental Copper as Co-Stressors – A Multigenerational Study

We examined the impacts of ocean acidification and copper as co-stressors on the reproduction and population level responses of the benthic copepod Tisbe battagliai across two generations. Naupliar production, growth, and cuticle elemental composition were determined for four pH values: 8.06 (control); 7.95; 7.82; 7.67, with copper addition to concentrations equivalent to those in benthic pore waters. An additive synergistic effect was observed; the decline in naupliar production was greater with added copper at decreasing pH than for decreasing pH alone. Naupliar production modelled for the two generations revealed a negative synergistic impact between ocean acidification and environmentally relevant copper concentrations. Conversely, copper addition enhanced copepod growth, with larger copepods produced at each pH compared to the impact of pH alone. Copepod digests revealed significantly reduced cuticle concentrations of sulphur, phosphorus and calcium under decreasing pH; further, copper uptake increased to toxic levels that lead to reduced naupliar production. These data suggest that ocean acidification will enhance copper bioavailability, resulting in larger, but less fecund individuals that may have an overall detrimental outcome for copepod populations.     

The effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on K+ transport,ATP level and intracellular pH of Chlorella fusca

1. Low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced net K⁺ uptake by Chlorella fusca, optimal concentrations being 3 μM CCCP in the light and 1 μM CCCP in the dark. Higher concentrations increasingly stimulated K⁺ release. 2. Measurements of the unidirectional K⁺ fluxes showed that CCCP-induced net K⁺ uptake in the light was mainly a consequence of an inhibition of efflux. In the dark, influx was slightly stimulated in addition. 3. In conditions of CCCP-induced net K⁺ uptake, the ATP level was decreased by less than 10%. With higher CCCP concentrations it fell drastically. 4. By means of the 5,5-dimethyloxazolidine-2,4-dione distribution technique, an acidification of the cell interior on the addition of CCCP was found. 5. It is concluded that uncoupler-induced net K⁺ uptake is due to an enhanced proton leakage into the cell across the plasmalemma. Intracellular acidification by this process stimulates ATP-dependent K⁺/H⁺ exchange which, in itself, is not affected at low uncoupler concentrations.     

Acidification and the structure of crustacean zooplankton in mountain lakes: The Tatra Mountains (Slovakia,Poland)

Species composition of planktonic Crustacea in 102 lakes in the West and High Tatra Mountains, studied during the peak of anthropogenic acidification (1978–1996), is presented in this work. Zooplankton of the Tatra lakes have been studied since the middle of the 19^th century, which later enabled the recognition of lake acidification and the assessment of its effect on the plankton community of lake ecosystems. In the pre-acidification period, the distribution of zooplankton was determined namely by the lake altitude and orientation (north vs. south) and by the catchment character. Crustacean zooplankton in larger lakes consisted of a limited number of species, with Acanthodiaptomus denticornis and Daphnia longispina dominating lakes in the forest zone, and Arctodiaptomus alpinus, Cyclops abyssorum, Daphnia longispina, Daphnia pulicaria, and Holopedium gibberum dominating lakes in the alpine zone. Ceriodaphnia quadrangula, Daphnia obtusa, Daphnia pulex, and Mixodiaptomus tatricus occurred in lakes with high concentrations of dissolved organic matter and in strongly acidified waters. Anthropogenic acidification has caused drastic changes in both the chemistry and biology of the Tatra lakes. Based on their status during the acidification peak, lakes were divided into three categories: non-acidified (with no change in the species composition of crustacean zooplankton due to the acidification), acidified (planktonic Crustacea disappeared in lakes with meadow-rocky catchments), and strongly acidified lakes where original Crustacea in meadow-rocky catchment lakes disappeared and were replaced by populations of the acid-tolerant littoral species Acanthocyclops vernalis, Chydorus sphaericus, and Eucyclops serrulatus. The acidification-induced processes of oligotrophication and toxicity of aluminium played a key role in the extinction of species. Despite the first signs of biological recovery observed in the early 2000s, acidification remains the most important factor governing the structure of plankton in the Tatra lakes.     

Difference in physiological responses of growth,photosynthesis and calcification of the coccolithophore Emiliania huxleyi to acidification by acid and CO2 enrichment

Ocean acidification, one of the great global environmental issues at present, is expected to result in serious damage on marine calcareous organisms such as corals and calcifying algae, which potentially release huge amounts of CO₂ from the ocean to the atmosphere. The coccolithophore, Emiliania huxleyi (Haptophyceae), which frequently produces blooms, has greatly contributed to the biological CO₂ pump. This study was aimed at analyzing effects of how E. huxleyi responds to acidification. Acidification was performed by two methods, namely by just adding HCl under bubbling ordinary air at 8.2–8.4, 7.6–7.8 and 7.1–7.3 (acidification by HCl) and by bubbling with ordinary air or with increased CO₂ concentration such as 406, 816 and 1,192 ppm that maintained pH of the medium at 8.0–8.3, 7.6–7.9 and 7.5–7.7 (acidification by CO₂ enrichment). As a result, cell growth and cellular calcification of E. huxleyi were strongly damaged by acidification by HCl, but not by acidification by CO₂ enrichment. The activities of photosystems such as F _v/F _m and ?PSII were not affected by any acidification conditions while photosynthetic O₂ evolution was slightly stimulated. A ⁴⁵Ca-radiotracer experiment revealed that Ca²⁺-uptake was strongly suppressed by acidification with HCl. This suppression recovered after increasing the dissolved inorganic carbon (DIC) concentration and further stimulated by an additional increase in DIC concentration. The production of storage and coccolith polysaccharides was increased by acidification by HCl and also highly stimulated by acidification with CO₂ enrichment. The present study clearly showed that the coccolithophore, E. huxleyi, has an ability to respond positively to acidification with CO₂ enrichment, but not just acidification.     

Effects of seawater acidification on Diopatra neapolitana (Polychaete,Onuphidae): Biochemical and regenerative capacity responses

Changes in atmospheric CO₂ concentrations and, consequently, ocean acidification, together with changes in seawater chemistry, are likely to have a large impact on marine ecosystems. Much of the current research concerning the consequences of ocean acidification has been limited to organisms dependent on the availability of carbonate ions in seawater, especially bivalves. Nevertheless, many marine organisms do not rely on calcium carbonate structures, such as most of the polychaete species that might also be affected by seawater acidification. However the effects of ocean acidification on these organisms are almost unknown. Thus, in the present study, the effects of pH decrease were studied in the polychaete Diopatra neapolitana, using tissue regenerative capacity and biochemical alterations as biomarkers. The results obtained revealed that individuals exposed for 28 days to low pH levels (7.5, 7.3, 7.1) exhibited lower capacity to regenerate their body, in comparison to control organisms (pH 7.8). This study also evidenced that seawater acidification induced oxidative stress in D. neapolitana, with individuals under lower pH levels showing higher LPO, lower GSH/GSSG values and higher enzymatic activity (CAT, SOD and GSTs). Additionally, organisms exposed to low pH presented significantly lower glycogen and protein content. Thus, the present work revealed the effects of pH on the polychaete species D. neapolitana, both at physiological and biochemical levels. Furthermore, our results validate the use of D. neapolitana as a test organism in laboratory-based bioassays, and also as an adequate bioindicator species to pH decrease in the environment. Finally, the regenerative capacity appears to be a promising endpoint to evaluate the effects of pH on D. neapolitana.     

Light-Induced Proton Release by the Cyanobacterium Anabaena variabilis: Dependence on CO(2) and Na

Light-induced acidification by the cyanobacterium Anabaena variabilis is biphasic (a fast phase I and slow phase II) and shown to be sodium-dependent with an optimum concentration of 40 to 60 millimolar Na⁺. Cells grown under low CO₂ concentrations at pH 9 (i.e. mainly HCO₃⁻ present in the medium) exhibited the slow phase II of proton efflux only, while cells grown under low CO₂ concentrations at pH 6.3 (i.e. CO₂ and HCO₃⁻ present) exhibited both phases. Light-induced proton release of phase I was dependent on inorganic carbon available in the bathing medium with an apparent K_m for CO₂ of 20 to 70 micromolar. As was concluded from the CO₂ dependence of acidification measured at different pH of the bathing medium, bicarbonate inhibited phase-I acidification noncompetetively. Acidification was inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Apparently, acidification of phase I is due to a light-dependent uptake of CO₂ being converted to HCO₃⁻ by a carbonic anhydrase-like function of the HCO₃⁻-transport system (M Volokita, D Zenvirth, A Kaplan, L Reinhold 1984 Plant Physiol 76: 599-602) before or during entering the cell, thus releasing one proton per CO₂ converted to HCO₃⁻.