Papers by Griselda Genovese
Marine and Freshwater Research
Context Bivalve fishery represents an important socio-economic resource in Río Negro province, an... more Context Bivalve fishery represents an important socio-economic resource in Río Negro province, and Mytilus platensis is the main exploited species. Thus, biomonitoring trace metals in this mussel is particularly relevant for both environmental protection and human consumption. Aim Anthropogenic pollution and its environmental impact on mussels were experimentally evaluated in situ in two coastline sites of northern Patagonia. Methods Trace metal concentrations were quantified in the water of a harbour area (Port of San Antonio Este) and a reference site (Isla Mejillón), an extraction location of M. platensis, were quantified. Metal accumulation and its effects on metabolic parameters were also measured in mussels. Results Higher concentrations of chromium, nickel and zinc in water were registered in the harbour, along with copper and chromium in gills, digestive gland and mantle of M. platensis, were registered in the harbour. Furthermore, superoxide dismutase activity was augmented...
General and Comparative Endocrinology, 2017
A game of two? Gene expression analysis of brain (cyp19a1b) and gonadal (cyp19a1a) aromatase in f... more A game of two? Gene expression analysis of brain (cyp19a1b) and gonadal (cyp19a1a) aromatase in females of a Neotropical cichlid fish through the parental care period and removal of the offspring, General and Comparative Endocrinology (2017),
Ecotoxicology and Environmental Safety, 2015
Endosulfan (ES) is an organochlorine pesticide widely used in agriculture despite its high toxici... more Endosulfan (ES) is an organochlorine pesticide widely used in agriculture despite its high toxicity towards non-target organisms such as fish. It has been demonstrated that ES can cause negative effects on aquatic animals, including disruption of hormonal systems. However, the alterations produced by this pesticide on the reproductive axis of fish prior to sexual maturity, as well as possible modes of action have hardly been studied. This study aimed at assessing the effect of waterborne exposure to the pesticide ES on the reproductive axis during sexual differentiation of juveniles of the South American freshwater cichlid fish Cichlasoma dimerus. No mortality was observed due to ES subchronic exposure (90 days post-fertilization). Exposure to ES did not affect body weight nor morphometric parameters, indicating that larvae nutritional state was not affected. Timing of sexual differentiation, gonadal morphology and sex ratio were likewise not altered by ES. However, ES acted as an endocrine disrupting chemical in this species as the morphometry of gonadotropin-releasing hormones (GnRH) producing cells was altered. Exposure to ES altered nuclear area, cell area and nucleus/cytoplasm ratio of GnRH II neurons, and cell and nuclear area and diameter of GnRH III neurons. Interestingly, in our previous study, exposure before sex differentiation (30 day exposure) caused no alteration to GnRH II and III, and did alter GnRH I and FSH cells. These alterations could lead to changes in circulating hormone levels, especially when fish are exposed for prolonged periods, ultimately impairing reproductive fitness. C. dimerus juveniles can be an interesting biological model to perform toxicological studies with the intent to assess early disruption endpoints in the reproductive axis during development.
Marine Pollution Bulletin
Pollution threats Antarctica and scientists blame xenobiotics and anthropic activities. Yet littl... more Pollution threats Antarctica and scientists blame xenobiotics and anthropic activities. Yet little is known about their effect on Antarctic ichthyofauna. Accordingly, we investigated the endocrine system of male fish Notothenia rossii and N. coriiceps (Perciformes, Nototheniidae) collected during Austral summer. For N. rossii, hormone levels of estradiol, testosterone, and 11-ketotestosterone were higher in fish collected during March than those of January; whereas for N. coriiceps estradiol and androgens levels were higher and lower in March, respectively. Histological analysis of the testes showed an unrestricted lobular type with no pathological alterations. However, detection of vitellogenin-like in plasma and skin mucus were seen in 75% of N. coriiceps males and 7% of N. rossii males. This is the first report of mucus vitellogenin-like detection as a non-invasive biomarker of endocrine disruption in notothenioid males and settles a baseline for future studies of reproductive biology and endocrine disruption in Antarctic fishes.
Polar Biology
Several fish species of the suborder Notothenioidei (Perciformes) predominate in the Antarctic Co... more Several fish species of the suborder Notothenioidei (Perciformes) predominate in the Antarctic Convergence Zone; nevertheless, reproductive studies are scarce due to difficulties on regular sampling. This study takes the research area of reproductive biology of notothenioids to a new level by providing, for the first time, data on sex hormone and vitellogenin detection in the blood of females of Notothenia rossii and N. coriiceps and correlates this data with morphological maturity indices as well as ovarian histology. Fish were captured during the Antarctic summer at Potter Cove, 25 de Mayo (King George) Island, and blood and ovary were collected. Histological analysis revealed that females of both fish possess group synchronous ovarian development with two distinct clutches of oocytes: a more advanced batch of vitellogenic oocytes ready for spawning and a second batch of previtellogenic oocytes for the next spawning event. Since liver vitellogenin synthesis is stimulated by estradiol produced by the ovaries, gonadal development, estradiol levels, and vitellogenin showed that both species were at a more advanced stage of maturation in March than in January. On the other hand and irrespectively of the month, gonadosomatic index and plasma estradiol levels of N. coriiceps were higher than those of N. rossii. Furthermore, females of N. coriiceps showed an advanced stage of vitellogenesis or were ready for spawn, contrary to results of previous studies. Our results indicate the successful use of gonadal morphology, estradiol, and vitellogenin detection for the estimation of sexual maturity stage of female adults.
Aquatic Toxicology, 2014
In oviparous vertebrates, vitellogenin (VTG) is mainly produced by the liver in response to estro... more In oviparous vertebrates, vitellogenin (VTG) is mainly produced by the liver in response to estrogen (E 2 ) and its synthesis is traditionally coupled to estrogen receptor alpha induction. Even though VTG is a female-specific protein, chemicals that mimic natural estrogens, known as xenoestrogens, can activate its expression in males causing endocrine disruption to wildlife and humans. Alkylphenols such as nonylphenol (NP) and octylphenol (OP) are industrial additives used in the manufacture of a wide variety of plastics and detergents, and can disrupt endocrine functions in exposed animals. For more than a decade, the freshwater cichlid fish Cichlasoma dimerus has been used for ecotoxicological studies in our laboratory. We recently found an up-regulation of VTG gene expression in livers of male fish exposed to OP, from a silent state to values similar to those of E 2 -induced fish. To better understand the underlying mechanisms behind the action of xenoestrogens, the aim of this study was to analyze the dose-response relationship of C. dimerus VTG and estrogen receptors (ERs) gene expression after waterborne exposure to 0.15, 1.5, 15, and 150 g/L OP for up to 1 month (0, 1, 3, 7, 14, 21, and 28 days). At the end of the experiment, histological features of exposed fish included active hepatocytes with basophilic cytoplasm and high eosinophilic content in their vascular system due to augmented expression of VTG. In testis, high preponderance of sperm was found in fish exposed to 150 g/L OP. A classic dose-response downregulation of the expression of Na + /K + -ATPase, a "non-gender specific gene" used for comparison, was found with increasing OP concentrations. No VTG and very low levels of ER␣ were detected in control male livers, but an up-regulation of both genes was found in males exposed to 0.15 or 150 g/L OP. Moreover, VTG transcripts were significant as early as day 3 or day 1 of exposure to these OP concentrations, respectively. Nearly no response was detected in 1.5 and 15 g/L OP exposed-fish. Data was curve-fitted evidencing a nonmonotonic dose-response curve. Interestingly, ER2 mRNA expression was augmented above baseline levels only when males were exposed to the lowest OP concentration. We speculate that genomic control of vitellogenesis is under control of multiple steroid receptors with different affinities for ligands. ER isoform, only up-regulated with very low concentrations of ligand, would act as a sensors of OP (or E 2 ) to induce ER␣ and VTG. With high OP concentrations, the expression of ER␣ isoform is promptly augmented, with the concomitant VTG transactivation.
Journal of Experimental Biology, 2006
Aquatic Toxicology, 2012
The aim of this study was to describe the time-course of estrogen-induced gene expression, corres... more The aim of this study was to describe the time-course of estrogen-induced gene expression, corresponding plasma protein detection and histological alterations after cessation of octylphenol (OP) exposure of Cichlasoma dimerus, to test differential responses of biomarkers suitable for environmental monitoring. Male fish were exposed to a nominal concentration of 150 g/L OP for 28 days, and later transferred to OP-free water aquaria for 1, 3, 7, 14, 21 or 28 days. Blood and mucus samples were obtained in order to analyze vitellogenin (VTG) and zona pellucida (ZP) proteins by Western blot; liver samples were used for gene expression and to assess tissue damage and further recovery of all the analyzed endpoints. Partial sequences of C. dimerus VTG and Na + /K + -ATPase were obtained. Comparison with VTGs of several teleosts supports that the partial sequence obtained for C. dimerus belongs to VTGAb type. ZP and VTG expression was highly up-regulated by OP. Immunoreactive (ir-) bands of 62, 52 and 50 kDa for ZP and 140, 103, 75 and 64 kDa for VTG, were detected after 28 days of OP exposure in plasma and mucus samples. After transfer of treated fish to clean water, ZP ir-bands in plasma disappeared rapidly (day 3), while VTG ir-bands decreased gradually; no ir-bands were detected on day 28 of recovery. Similarly, ZPB transcripts abruptly returned to background levels (day 3), earlier than for ZPC (day 7) or VTG (day 14). Liver from OP treated fish showed tissue disarrangement, eccentric and euchromatic hepatocytes nuclei and intense perinuclear basophilia. After the recovery period, these changes were still evident though less pronounced, accounting for irreversibility of tissue damage or the requirement for a longer period of depuration. The present results confirm that for biochemical and molecular biomarkers, such as induction of female proteins in male fish exposed to OP, complete recovery is achieved after adequate time of depuration (28 days). Male ZPB expression reflects a recent exposure to estrogenic contaminants, while VTG may reveal past exposures. The combination of biomarkers with different temporal responses such as C. dimerus ZP and VTG provides a more comprehensive interpretation of pollution status.
Aquatic Toxicology, 2011
An increasing number of widely used industrial and agricultural chemicals are being found to caus... more An increasing number of widely used industrial and agricultural chemicals are being found to cause endocrine disruption. In fishes, xenoestrogens can induce female proteins, and in some cases, the development of testis-ova, demonstrating feminization of males. In this study we analyzed the effect of an acute exposure of adult male Cichlasoma dimerus fish to estradiol (E 2 ) and octylphenol (OP). E 2 and OP were injected at 10 and 50 g/g body weight doses, respectively. After a single OP dose, liver was processed for RNA extraction at 1, 3, 12, 24, and 72 h. PCR was performed using cDNA and primers for egg coat or zona pellucida proteins (ZP). Genes encoding ZPB and ZPC isoforms were sequenced. E 2 -induced fish were sacrificed at 72 h. Using multiple OP or E 2 injections, blood and surface mucus were sampled on days 0, 3, 6, 9, and 13. On day 13 fish were sacrificed for liver and testis dissection. Histological examination of E 2 and OP-treated fish livers showed cellular disarray and intense cytoplasmatic basophilia within hepatocytes, probably due to increased mRNA synthesis, as well as hypertrophied euchromatic nuclei, and conspicuous nucleoli, indicative of augmented cell activity. An abnormal amount of sperm and immature germ cells within the testis lumen were seen in treated fish, suggesting reproductive impairment. Both plasma and mucus revealed the presence of ZP (and vitellogenin) at day 3 and thereafter with E 2 treatment, using Western and Dot blot techniques; OP effects were delayed in time. These results validate the analysis of mucus by Dot blot as an easy and rapid technique to address endocrine disruption caused by OP. Quantitative gene expression showed induction of liver ZPB and ZPC upon OP injection; muscle, brain, and intestine did not express any ZP. Both ZPs were induced at 1 h post injection, but only ZPB expression was statistically significant. At 12 h, both ZPs increased significantly, reaching the same levels of E 2 -challenged males after 72 h. Therefore, OP mimicked the action of E 2 with a prompt and strong xenoestrogenic effect, evidenced by the early response through mRNA and protein expression of ZP and the concomitant histological liver and testis alterations.
Ecotoxicology and Environmental Safety, 2015
Endosulfan (ES) is an organochlorine pesticide widely used in agriculture despite its high toxici... more Endosulfan (ES) is an organochlorine pesticide widely used in agriculture despite its high toxicity towards non-target organisms such as fish. It has been demonstrated that ES can cause negative effects on aquatic animals, including disruption of hormonal systems. However, the alterations produced by this pesticide on the reproductive axis of fish prior to sexual maturity, as well as possible modes of action have hardly been studied. This study aimed at assessing the effect of waterborne exposure to the pesticide ES on the reproductive axis during sexual differentiation of juveniles of the South American freshwater cichlid fish Cichlasoma dimerus. No mortality was observed due to ES subchronic exposure (90 days post-fertilization). Exposure to ES did not affect body weight nor morphometric parameters, indicating that larvae nutritional state was not affected. Timing of sexual differentiation, gonadal morphology and sex ratio were likewise not altered by ES. However, ES acted as an endocrine disrupting chemical in this species as the morphometry of gonadotropin-releasing hormones (GnRH) producing cells was altered. Exposure to ES altered nuclear area, cell area and nucleus/cytoplasm ratio of GnRH II neurons, and cell and nuclear area and diameter of GnRH III neurons. Interestingly, in our previous study, exposure before sex differentiation (30 day exposure) caused no alteration to GnRH II and III, and did alter GnRH I and FSH cells. These alterations could lead to changes in circulating hormone levels, especially when fish are exposed for prolonged periods, ultimately impairing reproductive fitness. C. dimerus juveniles can be an interesting biological model to perform toxicological studies with the intent to assess early disruption endpoints in the reproductive axis during development.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 1999
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2000
Oysters Crassostrea gigas originating from a metal-enriched estuary (Gironde estuary, France) wer... more Oysters Crassostrea gigas originating from a metal-enriched estuary (Gironde estuary, France) were translocated to a
Toxicology, 2006
The aim of this work was to study the transference of hexachlorobenzene from a green alga (Chlore... more The aim of this work was to study the transference of hexachlorobenzene from a green alga (Chlorella kessleri) to an estuary crab (Chasmagnathus granulatus), and to analyze the toxic effects that the xenobiotic has on the latter. The effect of hexachlorobenzene uptake was evaluated measuring oxidative stress, Uroporphyrinogen decarboxylase activity and morphometric parameter alteration, and also performing a histological analysis of crab hepatopancreas. Results demonstrated that hexachlorobenzene enters the alga, is accumulated in it, and then transferred into the crab, causing a decrease in Uroporphyrinogen decarboxylase activity in both organisms. The high malondialdehyde levels detected in crab hepatopancreas after the toxic treatment suggested the existence of hexachlorobenzene-induced lipid peroxidation. Antioxidant defenses such as superoxide dismutase activity and reduced glutathione content fell below normal values on the fourth week of treatment. At the same time, the hepatosomatic index, used as a morphometric parameter, reduced 20% with respect to the control. The histological analysis revealed epithelium disorganization in hepatopancreas tubules, confirming the existence of structural damage caused by hexachlorobenzene.
Toxicology, 2006
The aim of this work was to study the transference of hexachlorobenzene from a green alga (Chlore... more The aim of this work was to study the transference of hexachlorobenzene from a green alga (Chlorella kessleri) to an estuary crab (Chasmagnathus granulatus), and to analyze the toxic effects that the xenobiotic has on the latter. The effect of hexachlorobenzene uptake was evaluated measuring oxidative stress, Uroporphyrinogen decarboxylase activity and morphometric parameter alteration, and also performing a histological analysis of crab hepatopancreas. Results demonstrated that hexachlorobenzene enters the alga, is accumulated in it, and then transferred into the crab, causing a decrease in Uroporphyrinogen decarboxylase activity in both organisms. The high malondialdehyde levels detected in crab hepatopancreas after the toxic treatment suggested the existence of hexachlorobenzene-induced lipid peroxidation. Antioxidant defenses such as superoxide dismutase activity and reduced glutathione content fell below normal values on the fourth week of treatment. At the same time, the hepatosomatic index, used as a morphometric parameter, reduced 20% with respect to the control. The histological analysis revealed epithelium disorganization in hepatopancreas tubules, confirming the existence of structural damage caused by hexachlorobenzene.
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2004
Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills o... more Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10x salinity. Addition of cAMP agonists, such as cp-cAMP, forskolin, and IBMX, produced a significant increase in the transepithelial potential difference (Vte), which reflects ion transport activity. Dopamine (DA) also had a stimulatory effect on ion uptake, increasing Vte and Na + influx, although this effect was transient, since both variables remained elevated for less than 30 min. In addition, the dose-response curve for DA concentration-Vte was biphasic, and the maximum stimulation was obtained with 10 Amol l À1 . When the effects of forskolin and DA on the Na + /K + -ATPase activity were tested, they correlated well with the Vte and Na + influx experiments; the enzyme activity increased significantly after preincubation of gill fragments for 10 min with forskolin or DA (51 and 64%, respectively), but there was no effect after pre-incubation with DA for 20 min. Finally, KT5720, a specific inhibitor of cAMP-dependent protein kinase (PKA), completely abolished the stimulatory effect of DA on Vte, suggesting the involvement of PKA in this mechanism. D
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 1999
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2004
Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills o... more Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10x salinity. Addition of cAMP agonists, such as cp-cAMP, forskolin, and IBMX, produced a significant increase in the transepithelial potential difference (Vte), which reflects ion transport activity. Dopamine (DA) also had a stimulatory effect on ion uptake, increasing Vte and Na + influx, although this effect was transient, since both variables remained elevated for less than 30 min. In addition, the dose-response curve for DA concentration-Vte was biphasic, and the maximum stimulation was obtained with 10 Amol l À1 . When the effects of forskolin and DA on the Na + /K + -ATPase activity were tested, they correlated well with the Vte and Na + influx experiments; the enzyme activity increased significantly after preincubation of gill fragments for 10 min with forskolin or DA (51 and 64%, respectively), but there was no effect after pre-incubation with DA for 20 min. Finally, KT5720, a specific inhibitor of cAMP-dependent protein kinase (PKA), completely abolished the stimulatory effect of DA on Vte, suggesting the involvement of PKA in this mechanism. D
Crustaceana, 2000
Histological and morphometric analyses were performed on the gills of the semiterrestrial estuari... more Histological and morphometric analyses were performed on the gills of the semiterrestrial estuarine crab Chasmagnathus granulatus. Three different epithelia were recognized: (1) A thin epithelium, 2:42 § 0:33 ·m thick, which lines the whole lamellae in the three anterior gills and was assigned to respiratory functions. (2) A thick cuboidal epithelium, 6-12.5 ·m thick, covering part of gills 4 and 5 and the better part of the three most posterior gills. This tissue seems to be involved in ion-regulation, since it is located in the same zones which are darkly stained with silver nitrate and possess large numbers of mitochondria, closely associated to basolateral interdigitations and abundant infoldings of the apical membrane. (3) An attenuated epithelium, 0.5-1.5 ·m thick, bordering the marginal channels of all the gills. This tissue is clearly of a respiratory type and probably plays an important role during air breathing when the branchial water stores become reduced by evaporation, and the consequent gill collapse impairs both ventilation and perfusion of the central part of the lamellae.
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Papers by Griselda Genovese