Adriana Cristina Cochón

Inhibition of cholinesterases and carboxylesterases of two invertebrate species, Biomphalaria glabrata and Lumbriculus variegatus, by the carbamate pesticide carbaryl.

In this study, the effects of sublethal concentrations of the carbamate carbaryl on the cholinesterase (ChE) and carboxylesterase (CES) activities present in the oligochaete Lumbriculus variegatus and in the pigmented Biomphalaria glabrata gastropod were investigated. The results showed that ChE activity from both species was inhibited by in vivo and in vitro exposure to carbaryl, with EC(50) and IC(50) values approximately 20 times lower for the oligochaete than for the gastropod. On the other hand, the recovery process in uncontaminated media was more efficient in oligochaetes than in snails. Thus, in only 2h the oligochaetes showed no inhibition with respect to control values whereas the snails did not reach control values even after 48h of being in pesticide-free water. CES activity was investigated in whole body soft tissue homogenates using three different substrates: p-nitrophenyl butyrate, 1-naphthyl acetate (NA) and 2-NA. In addition, the presence of multiple CES isozymes in L. variegatus and B. glabrata extracts, with activity towards 1- and 2-NA, was confirmed by native polyacrylamide electrophoresis. In both species, the activities measured using the naphthyl substrates were higher than the activity towards p-nitrophenyl butyrate. In addition, B. glabrata showed a higher CES activity than L. variegatus independently of the substrate used. In L. variegatus, in vivo CES activity towards the different substrates was less sensitive to carbaryl inhibition than ChE activity. In contrast, in B. glabrata, CES activity towards p-nitrophenyl butyrate was inhibited at lower insecticide concentrations than ChE. The results of this study contribute to the knowledge of the sensitivity of non-target freshwater invertebrate Type B-esterases towards pesticides.

Effects of azinphos-methyl exposure on enzymatic and non-enzymatic antioxidant defenses in Biomphalaria glabrata and Lumbriculus variegatus.

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The oligochaete Lumbriculus variegatus and pigmented and non-pigmented specimens of the gastropod Biomphalaria glabrata are freshwater invertebrates that have been recommended for contamination studies. Recently, it has been shown that L. variegatus worms exhibit a higher cholinesterase (ChE) activity and a greater sensitivity to in vivo ChE inhibition by azinphos-methyl than pigmented B. glabrata snails. The aims of the present study were (1) to investigate if, in addition to its anticholinesterase action, azinphos-methyl has also pro-oxidant activity in L. variegatus and B. glabrata, and (2) to examine if species that are highly susceptible to the neurotoxic effects of organophosphates also suffer a greater degree of oxidative stress. Therefore, total glutathione (t-GSH) levels and activities of cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PDH) were measured in the whole body soft tissue of organisms exposed for 48 and 96 h to a level of azinphos-methyl that produces 50% of inhibition on ChE. Results showed different patterns of antioxidant responses between the gastropods and the oligochaetes, and even between the two phenotypes of gastropods: (1) in exposed L. variegatus t-GSH levels increased and CAT and SOD activities decreased with respect to control organisms, (2) in pigmented gastropods, SOD decreased while CAT transiently diminished, and (3) in non-pigmented gastropods, SOD activity showed a biphasic response. GST and G6PDH were not altered by azinphos-methyl exposure. Of note, t-GSH levels were 4-fold times higher in L. variegatus than in both phenotypes of B. glabrata. This may suggest that GSH could play a more important role in antioxidant defense in L. variegatus than in B. glabrata.

Cholinesterase and carboxylesterase inhibition in Planorbarius corneus exposed to binary mixtures of azinphos-methyl and chlorpyrifos.

Though pesticide mixtures are commonly encountered in fresh water systems, the knowledge of their effects on non-target aquatic species is scarce. The present investigation was undertaken to explore the in vivo inhibition of the freshwater gastropod snail Planorbarius corneus cholinesterase (ChE) and carboxylesterases (CES) activities by the organophosphorus pesticides azinphos-methyl (AZM) and chlorpyrifos (CPF). To this end, snails were exposed for 48 h to different concentrations of AZM and CPF in single-chemical and binary-mixture studies, and ChE and CES activities were measured in the whole soft body tissues and hemolymph. ChE activity was measured with acetylthiocholine as substrate and CES activity was measured with four substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1- and 2-naphthyl acetate. Single-chemical experiments showed that CPF was a more potent inhibitor of ChE activity than AZM (350 and 27 times for the whole soft tissue and hemolymph, respectively). CES were 15 times more sensitive than ChE when the activities were measured in the whole soft tissue of the animals exposed to AZM. Based on a default assumption of concentration addition, P. corneus snails were exposed to mixtures of AZM+CPF designed to yield predicted soft tissue ChE inhibitions of 31% (mixture 1), 50% (mixture 2) and 61% (mixture 3). Results showed that ChE inhibition produced by mixture 1 followed a model of concentration addition. In contrast, the other mixtures showed synergism, both in whole soft tissue and hemolymph. In addition, results obtained when the snails were exposed sequentially to the pesticides showed that the sequence AZM/CPF produced at 48 h a higher ChE inhibition than the sequence CPF/CPF. A range of metabolic pathways and responses associated with bioactivation or detoxification may play important roles in organophosphorus interactions. In particular, the data presented in the present study indicate that CES enzymes would be important factors in determining the effects of the mixtures of OPs on P. corneus ChE activity.

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus.

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary combinations of AZM-oxon and CPF-oxon caused a synergistic effect on the ChE inhibition in P. corneus homogenates. The degree of synergism tended to increase as the ratio of AZM-oxon to CPF-oxon decreased. These results suggest that synergism is likely to occur in P. corneus snails exposed in vivo to binary mixtures of the OPs AZM and CPF.

Effects of the organophosphate insecticide azinphos-methyl on the reproduction and cholinesterase activity of Biomphalaria glabrata.

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The snail Biomphalaria glabrata is a freshwater gastropod widely distributed in South America, Central America and Africa. The aim of the present work was to investigate whether azinphos-methyl causes alterations in the reproduction of B. glabrata. To this end, gastropod pigmented specimens were exposed to various concentrations of the insecticide (0.021, 0.5, 2.5, and 5 mg L(-1)) for either 2 or 14 d. Along 14 d, several reproduction parameters and cholinesterase (ChE) activity were evaluated. In each group, the number of egg masses, the number of eggs per mass, the number of hatchings, the time to hatching, and the survival of the offspring after one month of treatment was evaluated. The results showed that, depending on the concentration and time of exposure, azinphos-methyl induced alterations in the reproduction of B. glabrata. These alterations were mainly represented by a decrease in the number of egg masses, and, in some cases, by a lower number or even the total absence of hatchings. Thus, the gastropods exposed to 2.5 and 5 mg L(-1) of azinphos-methyl for 14 d showed ChE inhibitions higher than 35% along time and completely lost their ability to reproduce. On the other hand, exposure to high acute concentrations or exposure to low concentrations for 14 d resulted in ChE inhibition equal to or lower than 35% between 7 and 14 d of treatment and similar alterations in reproduction. These were represented by a decrease in the number of egg masses. At low pestice levels, the number of egg masses and the number of offspring resulted to be more sensitive biomarkers than ChE inhibition. It is concluded that the insecticide azinphos-methyl can cause a decline in the reproductive performance of B. glabrata.

Evaluation of the Porphyrinogenic Risk of Antineoplastics

The use of antineoplastics is common in cancer therapy, and some of them have been associated with the development of porphyria in patients with cancer. However, knowledge of their effects on the haeme metabolic pathway is at present scarce and unclear. So, the present study evaluates the porphyrinogenic ability of nine antineoplastics (both alkylating and non‐alkylating). These were tested either alone or in conjunction with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (latent porphyria model) in chick embryos and in mice. The results obtained suggest that the use of cyclophosphamide, azathioprine, 5‐fluorouracil, busulphan, procarbazine and hexamethylmelamine be avoided in the treatment of porphyric patients. On the other hand, dacarbazine, chlorambucil and melphalan are non‐porphyrinogenic. We also provide evidence showing that neither the presence of the mustard group in the structure of the antineoplastic nor alterations in ferrochelatase or protoporphyrinogen oxidase activities are responsible for the porphyrinogenic ability of cyclophosphamide.

In vivo studies on inhibition and recovery of B-esterase activities in Biomphalaria glabrata exposed to azinphos-methyl: Analysis of enzyme, substrate and tissue dependence

Cholinesterases and carboxylesterases belong to the group of B-esterases, the serine superfamily of esterases that are inhibited by organophosphorus compounds. It is now generally accepted that before using the B-esterases as biomarkers of exposure to organophosphorus and carbamates in a given species, the biochemical characteristics of these enzymes should be carefully studied. In this study, the enzyme/s and the tissue/s to be selected as sensitive biomarkers of organophosphorus exposition in the freshwater gastropod Biomphalaria glabrata were investigated. Firstly, the substrate dependence of cholinesterase and carboxylesterase activities in whole organism soft tissue and in different tissues of the snail (head-foot, pulmonary region, digestive gland, and gonads) was analyzed. Measurements of cholinesterase activity were performed using three substrates: acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), and butyrylthiocholine (BuSCh). Carboxylesterase activity was determined using four different substrates: 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). Regardless of the tissue analyzed, the highest specific activity was obtained when using AcSCh, followed by PrSCh. Cholinesterase activity measured with BuSCh was very low in all cases. On the other hand, the highest cholinesterase activity was measured in head-foot and in pulmonary region, representing in the case of AcSCh hydrolysis 196% and 180% of the activity measured in whole organism soft tissue, respectively. In contrast, AcSCh hydrolysis in digestive gland and gonads was 28% and 50% of that measured in whole organism soft tissue. Regarding carboxylesterase activity, although all tissues hydrolyzed the four substrates assayed, substrate preferences varied among tissues. In particular, digestive glands showed higher carboxylesterase activity than the other tissues (299%, 359% and 137% of whole organism soft tissue activity) when measured with 1-NA, 2-NA and p-NPA as substrates, respectively. In contrast, with p-NPB as substrate, the highest carboxylesterase activity was observed in pulmonary region. Exposure of the snails for 48 h to azinphos-methyl concentrations in the range of 0.05-2.5 mg L⁻¹ resulted in different degrees of inhibition of cholinesterase and carboxylesterase activities, depending on the enzyme, pesticide concentration, the substrate, and the tissue analyzed. In general, carboxylesterase activity measured with p-NPA and p-NPB was much more sensitive to azinphos-methyl inhibition than cholinesterase activity. The results also showed that while B-esterase activities in whole organism soft tissue and pulmonary region recovered completely within 14 days, carboxylesterase activity in digestive glands remained highly inhibited. On the whole, the results of the present study emphasize how important it is to characterize and measure cholinesterase and carboxylesterase activities jointly to make a proper assessment of the impact of organophosphorus pesticides in non-target species.

Azinphos-methyl and chlorpyrifos, alone or in a binary mixture, produce oxidative stress and lipid peroxidation in the freshwater gastropod Planorbarius corneus

Azinphos-methyl (AZM) and chlorpyrifos (CPF) are broad-spectrum organophosphate insecticides used for pest control on a number of food crops in many parts of the world that have been shown to inhibit cholinesterase activity in the non-target freshwater gastropod Planorbarius corneus. The present study was undertaken to determine: (a) whether AZM and CPF induce oxidative stress in P. corneus, and (b) whether a mixture of both organophosphates that causes a higher neurotoxicity than single pesticides also causes an enhanced oxidative stress. To this end, non-enzymatic and enzymatic parameters were measured in the soft tissues of snails acutely exposed to the insecticides in single-chemical (2.5 mg AZM L(-1) and 7.5 μg CPF L(-1)) and a binary-mixture (1.25 mg AZM L(-1) plus 3.75 μg CPF L(-1)) studies. At 24 h, all pesticide-exposed groups showed significantly decreased glutathione (GSH) and glutathione disulfide (GSSG) levels when compared to control animals. At 48 h, all exposed groups showed an alteration of the redox status (GSH/GSSG ratio) and a significant increase in malondialdehyde levels. The exposure for 48 h to AZM and CPF, alone or in the binary mixture, also resulted in a significant decrease of the antioxidant superoxide dismutase activity. The greatest decrease was observed with CPF exposure (59% of decrease relative to the control group). A significant increase in catalase and glutathione S-transferase activities was observed in CPF group and in CPF and AZM+CPF groups, respectively. The activities of glutathione reductase and glucose 6-phosphate dehydrogenase did not show significant changes with respect to controls in any treatment group. In conclusion, the data shown in the present study provide evidence that AZM, CPF and a mixture of both organophosphates are able to induce oxidative stress and oxidative damage in P. corneus tissues. However, no similarities between the degree of neurotoxicity and the degree of alterations of the measured oxidative stress parameters were found.

Effects of the porphyrinogenic compounds hexachlorobenzene and 3,5-diethoxycarbonyl-1,4-dihydrocollidine on polyamine metabolism

The naturally occurring polyamines--putrescine, spermidine and spermine--are organic cations present in all living cells and essential for cell growth and differentiation. The aim of the present study was to extend the investigations on the effects of porphyrinogenic compounds on polyamine metabolism. This was achieved by studying putrescine, spermidine and spermine levels in a model of acute porphyria, i.e. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced porphyria, and in a model of non-acute porphyria, i.e. hexachlorobenzene (HCB)-induced porphyria. HCB administration to female Wistar rats for 7, 14, 21, 28 and 56 days did not alter polyamine levels in liver, even though rats presented clear signs of HCB-induced porphyria. In contrast to HCB, DDC treatment resulted in a remarkable increase in putrescine levels in the liver of female and male Sprague-Dawley rats. This increase was due, at least in part, to ornithine decarboxylase (ODC) activation. DDC induction of putrescine levels did not show organ specificity, since it could also be seen in adrenal gland. Interestingly, the deregulation of polyamine biosynthesis occurred concomitantly with the deregulation of the heme biosynthetic pathway. In addition to porphyria, it is known that DDC intoxication affects several proteins of the hepatocyte cytoskeleton. It is suggested that DDC-induced increase in ODC activity and putrescine levels may be an early event contributing to alter the cytoskeleton.

Phospholipid alterations elicited by hexachlorobenzene in rat brain are strain-dependent and porphyria-independent.

Hexachlorobenzene (HCB) alters phospholipid and heme metabolisms in the liver and Harderian gland. The effects of HCB on phospholipid metabolism, in an organ considered to be non-responsive to its porphyrinogenic effects, remain to be studied. Therefore, as the brain is an organ with this feature, this paper analyzes the effects of HCB on brain phospholipid composition in order to investigate if there is any relationship between HCB-induced porphyrin metabolism disruption and phospholipid alterations. For this purpose, a time-course study of HCB effects on brain phospholipids was performed in two strains of rats differing in their susceptibility to acquire hepatic porphyria: Chbb THOM (low); and Wistar (high). This paper shows for the first time that rat brain phospholipids are affected by HCB exposure. Comparative studies show that HCB-induced disturbances in brain phospholipid patterns are time and strain-dependent. Thus, whereas major phospholipids, phosphatidylcholine and phosphatidylethanolamine were more altered in Wistar rats, minor phospholipids, phosphatidylinositol and phosphatidylserine were more affected in Chbb THOM rats. HCB intoxication led to a sphingomyelin/phosphatidylcholine molar ratio lower than the normal, in both strains. As was expected, brain porphyrin content was not altered by HCB intoxication in either strain. It can be concluded that HCB is able to alter brain phospholipid metabolism in a strain-dependent fashion, and in the absence of alterations in brain heme metabolism. In addition, HCB-induced disturbances in brain phospholipids were not related to the degree of hepatic porphyria achieved by the rats.