**5. Carbonic anhydrase and heavy metals interactions: Potential applications**

In the last years the interactions between carbonic anhydrase and heavy metals have found a number of applications in environmental and health fields, including the development of biomarkers of pollution exposure, in vitro bioassays, and biosensors.

#### **5.1 Carbonic anhydrase sensitivity to heavy metals and development of biomarkers of pollution exposure**

Pollution by trace metals is a world-wide problem due to the persistency and continuing accumulation of metals in the environment (de Mora et al. 2004; Hwang et al 2006). Heavy metals may enter the organisms through food, water, air, or absorption through the skin. As a result of mining, waste disposal and fuel combustion the environment is becoming increasingly contaminated with heavy metals.

In recent years the increasing sensibility to pollution problems has promoted the development of environmental "diagnostic" tools for early warning detection of pollution. Pollution monitoring has been increasingly concerned with the use of biological responses to pollutants at molecular and cellular level for evaluating biological hazard of toxic chemicals. Methods based on biological effects and their underlying mechanisms can complement the use of analytical chemistry in environmental monitoring. The major advantages of such biological, mechanism-based methods are their toxicological specificity, rapidity, and low cost. Toxicological specificity refers to the relationship between the assay response and the toxic potential rather than simply the contaminant concentrations (provided by chemical analysis) of the sample being analyzed. Moreover, biological assays provide rapid, sensitive, easily learnt and readily interpretable new useful tools for environmental biomonitoring and risk assessment. They include biomarkers, and *in vivo* and *in vitro* bioassays. It is known that the harmful effects of pollutants are typically first manifested at lower levels of biological organization before disturbances are realized at

al., 1994; Lee et al., 1995) are the only other examples reported in nature of CA activity increase induced by cadmium exposure. Evidence of *in vivo* utilization of Cd in CA has been found in microalgae (Price and Morel, 1990; Morel et al., 1994; Lee et al. 1995, Xu et al., 2008). In these organisms the ability of Cd to substitute for Zn at the active site of the enzyme is reflected in the regulation of the enzyme expression. In *Thalassiosira weissflogii* a cadmium-containing CA was found to be expressed during zinc limitation (Lane and Morel, 2000; Lane et al., 2005). This cadmium CA (CDCA1) which naturally uses Cd as its catalytic metal (Trip et al., 2001; Lane et al., 2005) has been ascribed to a novel ζ-CA class (see above). Genes coding for similar proteins have been identified in other cultured diatoms (Park et al., 2007). In mussel digestive gland western blotting analysis clearly demonstrated the enhancement of CA protein expression following cadmium exposure, according to the enzymatic activity data (Caricato et al., 2010). Laboratory experimental results were confirmed by a field experiment. Mussels exposed for 30 days to an anthropogenic impacted site showed a significant increase in CA activity and protein expression with respect to animals exposed for 30 days in a control site. If the new synthesized enzyme is a Cd-CA is not possible to say at the moment. If it was the case, then the increase in CA would not be a direct adaptive response to Cd pollution; rather, Cd could remove any limitations placed on CA synthesis by the availability of Zn. However*,* 

future studies will be needed to clarify this intriguing aspect of the research.

biomarkers of pollution exposure, in vitro bioassays, and biosensors.

**pollution exposure** 

increasingly contaminated with heavy metals.

**5. Carbonic anhydrase and heavy metals interactions: Potential applications**  In the last years the interactions between carbonic anhydrase and heavy metals have found a number of applications in environmental and health fields, including the development of

**5.1 Carbonic anhydrase sensitivity to heavy metals and development of biomarkers of** 

Pollution by trace metals is a world-wide problem due to the persistency and continuing accumulation of metals in the environment (de Mora et al. 2004; Hwang et al 2006). Heavy metals may enter the organisms through food, water, air, or absorption through the skin. As a result of mining, waste disposal and fuel combustion the environment is becoming

In recent years the increasing sensibility to pollution problems has promoted the development of environmental "diagnostic" tools for early warning detection of pollution. Pollution monitoring has been increasingly concerned with the use of biological responses to pollutants at molecular and cellular level for evaluating biological hazard of toxic chemicals. Methods based on biological effects and their underlying mechanisms can complement the use of analytical chemistry in environmental monitoring. The major advantages of such biological, mechanism-based methods are their toxicological specificity, rapidity, and low cost. Toxicological specificity refers to the relationship between the assay response and the toxic potential rather than simply the contaminant concentrations (provided by chemical analysis) of the sample being analyzed. Moreover, biological assays provide rapid, sensitive, easily learnt and readily interpretable new useful tools for environmental biomonitoring and risk assessment. They include biomarkers, and *in vivo* and *in vitro* bioassays. It is known that the harmful effects of pollutants are typically first manifested at lower levels of biological organization before disturbances are realized at population, community and ecosystem levels (Adams, 1990). This is the reason why in recent years the study of molecular and cellular effects of pollutants has given important advancement in the developing of biologically-based methodologies useful for environmental biomonitoring and risk assessment. Enzymatic inhibition studies have been a very fruitful field for environmental monitoring application as biomarker of exposure/effect. Biomarkers are defined as pollutant induced variation in cellular or biochemical components occurring in organisms as a result of natural exposure to contaminants in their environment (Depledge, 1994). As reported by several authors, the evaluation of biomarkers in bioindicator organisms sampled in one or more areas suspected of chemical contamination and their comparison with organisms sampled in a control area can allow the evaluation of the potential risk of toxicological exposure of the studied community (Lionetto et al., 2003; Lionetto et al., 2004).

Carbonic anhydrase sensitivity to heavy metal exposure has been recently explored for its possible applications as biomarker of exposure to heavy metal pollution (Lionetto et al. 2006; Caricato et al, 2010b.) in "sentinel" organisms. Lionetto et al., (2006) investigated CA activity inhibition by heavy metals in the filter feeding *Mytilus galloprovincialis*, widely used in pollution monitoring programs as sentinel organism (Jernelov et al., 1996). Following *in vitro* and *in vivo* exposure to cadmium, mantle CA activity was significant inhibited. The inhibitory effect of cadmium on mantle CA activity can explain results previously obtained by Soto et al. (2000), who observed a significant decreased in shell growth in *M. galloprovincialis* exposed to heavy metals. The sensitivity of CA to heavy metals in mussels appears to be tissue-specific. In fact, as reported above, in mussel's digestive gland CA activity and expression was found to increase following Cd exposure (Caricato et al., 2010). Because of the widely application of *M. galloprovincialis* in environmental quality monitoring and assessment, data on tissue specific sensitivity of carbonic anhydrase to heavy metals represent a starting point for future potential application of CA activity changes as biomarker of exposure to heavy metals in the sentinel organism *M. galloprovincialis*.

Other studies carried out on corals have suggested alteration in CA activity as potential biomarker of exposure to environmental chemical stress. CA activity has been demonstrated to be inhibited by heavy metal exposure in anemones and corals (Gilbert and Guzman, 2001), where the enzyme plays a key role in the calcification process. Coral growth has been shown to be an effective indicator of the overall health of a coral reef ecosystem and reduced growth can reflect impaired photosynthetic output of the zooxanthellae and/or changes in enzyme activity (Moya et al., 2008). In an era of climate change and ocean acidification, where factors impacting growth and resilience factors are becoming important, understanding the biological effects of metal exposure to these keystone tropical organisms may be critical (Bielmyer et al., 2010).

#### **5.2 Carbonic anhydrase based bioassay**

Bioassays use biological systems to detect the presence of toxic chemicals in the environmental matrices (water, sediment, sewage, soil, etc.). In recent years, *in vitro* bioassays, employing cultured cells or cellular extracts, are increasingly being developed and used to detect the presence of contaminants. Examples include assays that measure enzyme inhibition, receptor-binding, or changes in gene expression in *in vitro* systems. Although *in vitro* assay is not a substitute for biomarker approach, it can be used as an adjunct model to whole-animal *in vivo* exposure and to ecotoxicological evaluation of the potential risk of trace pollutants in aquatic environments. They are rapid, low cost and simple tools to be utilized in combination with chemical analysis, for the pre-screening of the environmental samples that should be analyzed. Lionetto et al (2005; 2006) explored the possible application of heavy metal CA inhibition for the development of an *in vitro* bioassay applicable to the determination of the toxicity of environmental aqueous samples. They developed rapid and sensitive chemical hazard detection system for standardizing rapid, sensitive, and low cost CA based *in vitro* bioassay (Schettino et al., 2008).
