Biliary and hepatic metallothionein, metals and trace elements in environmentally exposed neotropical cichlids Geophagus brasiliensis.

One of the metal detoxifying mechanisms that occurs in fish is metallothionein (MT) induction and metal binding. Hepatic MT induction has been well described, but biliary MT metal detoxification has only recently been described in fish. In this scenario, metal-metal interactions have been increasingly evaluated to further understand the behavior of these contaminants regarding homeostasis and biological functions, as well as their toxic effects. Studies, however, have been mainly conducted concerning the elemental pair Se-Hg, and scarce reports are available concerning other metal pairs. Therefore, this study aimed to evaluate biliary and hepatic MT metal detoxification mechanisms in a territorial neotropical cichlid, Geophagus brasiliensis. Fish were sampled from the anthropogenically impacted estuarine Rodrigo de Freitas Lagoon, located in Southern Rio de Janeiro, and trace elements and MT were determined by inductively coupled plasma mass spectrometry (ICP-MS) and UV-Vis spectrophotometry, respectively, in fish liver and bile. MT in bile were significantly lower than in liver. Significant differences between bile and liver were observed for many trace elements, and, although most were higher in liver, Cd and Ni were significantly higher in bile, indicating efficient excretion from the body via the biliary route. A significant correlation was observed between MT and Fe in bile, and between MT in liver and Cu and Zn in bile. Molar ratio calculations demonstrated protective elements effects against Al, As, Cd, Hg, Pb and V in both bile and liver, as well as some novel interrelationships, indicating the importance of these investigations regarding the elucidation of element detoxifying mechanisms. Furthermore, investigation of other elemental associations may aid in decision-making processes regarding environmental contamination scenarios linked to public health.

Bile and liver metallothionein behavior in copper-exposed fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

Evaluation and standardization of different purification procedures for fish bile and liver metallothionein quantification by spectrophotometry and SDS-PAGE analyses.

Fish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, ß-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70 °C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix.

Changes and variations of polycyclic aromatic hydrocarbon concentrations in fish, barnacles and crabs following an oil spill in a mangrove of Guanabara Bay, Southeast Brazil.

On April 26th, 2005, an accident caused a leak of 60,000L of Diesel Oil Type "B", freighted by train wagons upstream on a mangrove area within Guanabara Bay, Southeast Brazil. After the accident, samples from animals with different biological requirements were collected in order to monitor polycyclic aromatic hydrocarbons concentrations for the following 12months. Sessile, mobile, carnivorous, omnivorous, organic detritus feeders, planktivorous and suspension feeders were some of the attributes compared. Concentrations of PAHs did not vary in relation to different dietary habits and the best response was from the sessile suspensivorous barnacles. A background level of <50microgkg(-1) was suggested based on the reference site and on values observed in the following months after the accident. The highest values of PAH concentrations were observed in barnacles in the first month immediately after the spill, decreasing to background levels after few months. Barnacles are suggested as a sentinel species.

Operational problems related to the preparation of the seawater soluble fraction of crude oil.

Owing to the importance of dissolution and weathering processes following oil spills, this work focused on the operational (quantitative) aspects related to the dissolution of petroleum-derived products, as well as the influence of solar light on both dissolution and the photoproduction of hydrogen peroxide. Four Brazilian crude oil samples were used to study the transfer process of organic compounds from the crude oil film to the aqueous phase (natural seawater) over a period of up to 45 days. Dissolved organic carbon (DOC), measured by non-dispersive infrared spectroscopy followed by high temperature catalytic combustion, was used to follow the partitioning between the two phases. Aqueous DOC values increased as a function of time (up to 15 days) until equilibrium was reached at concentrations ranging from 5 to 45 mg C L(-1). The final DOC concentration as well as the rate of dissolution depends on the nature of the crude oil. When exposed to sunlight, the dissolution was enhanced by up to 67.3%, and inorganic peroxides were generated in the concentration range from 4.5 up to 8.0 micromol L(-1) after 7.3 h irradiation. These results indicate that there is a need for a standard procedure for the production of the WSF in order to generate a more reliable tool to assess the impact of oil spills on the marine environment.