**4. Heavy metals as modulators of carbonic anhydrase activity and expression**

If it has been widely demonstrated *in vitro* that heavy metals are able to inhibit CA activity in a variety of organisms, on the contrary little is known about the *in vivo* effects of trace metals on the activity and the expression of this metalloenzyme. The major information regards Zn2+, while very few is known about other metals.

In humans early studies demonstrated that dietary zinc deficiency significantly reduces zinc concentrations of serum and in turn CA activity in erythrocytes (Hove,1940; Rahman et al., 1961; Kirchgessner et al., 1975) suggesting a possible influence of Zn2+ on CA protein expression. These early data have been more recently confirmed by Lukaski (2005) who demonstrated zinc concentration of serum and erythrocyte to be positively correlated to CA activity *in vivo*. Low dietary zinc decreases erythrocyte carbonic anhydrase activity and, in turn, impairs cardiorespiratory function in men during exercise (Lukaski et al., 2005). In ducks Zn2+ at a low level (up to 1.25 μM Zn) induced the rise of CA activity in erythrocytes (Wu et al., 2007). In parotid saliva of patients with CAVI deficiency Zn2+ treatment was able to stimulate synthesis/secretion of CAVI (Henkin et al., 1999), probably through stimulation of CAIV gene. In rats Zn2+ deficiency *s*ignificantly reduced CAII protein expression in the submandibular gland (Goto et al., 2008).

As regards other metals Grimes et al (1997) reported the depression of CAIII mRNA and, in turn, CAIII protein in the mouse mutant 'toxic milk' (tx) liver following copper accumulation, Kuhara et al (2011) found CAIII suppression by copper accumulation during carcinogenesis, while Wu et al (2007) found iron at low levels to induce a rise in CA activity in duck erythrocytes.

Recently, Caricato et al (2010) demonstrated for the first time CA activity and protein expression to be enhanced by the exposure to the trace element cadmium in animals, opening new perspective in the comprehension of the functioning and regulation of this enzyme. Digestive gland CA activity showed a weak sensitivity to *in vitro* cadmium exposure since only high concentrations of CdCl2 (from 10-5 to 10-3 M) were able to exert a significant inhibition. On the contrary digestive gland CA activity showed a significant increment in cadmium exposed animals (about 40% after two week of exposure). This was the first time that CA activity appears to be increased by cadmium in animals. Carbonic anhydrases from the microalgae *Chlamydomonas reinhardtii* (Wang at al., 2005) and *Thalassiosira weissflogii* (Morel et 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.
