The blood clams, T. granosa, is an economically and ecologically significant bivalve species along China coastal waters. The demersal lifestyle makes them more susceptible to Bap pollution which are prone to be deposited in the sediment. However, there are very few studies on the effects of Bap on blood clams. Su et al. (2017) investigated the immunotoxicity induced by Bap under ocean acidification conditions, which is the only searchable study on the Bap toxicity on blood clams to our knowledge. Here, we performed acute Bap exposure on blood clams, and then assessed the effects of Bap toxicity: histological changes, oxidative stress, neurotoxicity and global DNA methylation.
As filter-feeding organisms, the gill tissues of molluscs are exposed to the external environment and function as the main entrance for waterborne toxicants, exerting the first environment-tissue barrier against contaminants (Luckenbach et al., 2008). Aiming to provide an intuitive understanding on the effects of Bap on the gills of blood clams, we investigated the histological changes in gills by means of H.E staining. Upon Bap exposure, some significant morphological abnormalities including gill filaments denuded of cilia, hemocyte infiltration and consequent breakage and necrosis, were observed. Similarly, in mussels M. galloprovincialis from the petrochemical polluted site, severe morphological alterations were also observed in gills (Maisano et al., 2017). These findings indicated that petrochemical pollutants represented by Bap can indeed cause damage to the gill structural organization of bivalve molluscs. Due to the important physiological significance of mussel gills in feeding efficiency and gas exchange (Sunila, 1988), the destruction of this structural organization may affect the functional integrity of the gills (Cappello et al., 2013).
Oxidative stress refers to an increase in intracellular ROS levels that leads to damage to lipids, proteins and DNA (Schieber and Chandel, 2014). It has been demonstrated as a primary Bap-mediated mechanism of toxicity in the molluscs (Livingstone, 2001). Here, we investigated the Bap induced oxidative stress as manifested by the change of antioxidative enzyme activities, LPO level and oxidative DNA damage in blood clams’ gills. No accidentally, SOD, CAT, POD and GST activities increased significantly after Bap exposure, just as has been demonstrated in numbers of molluscs. Despite of these, the fluctuation trends of enzymic activities were varied in the present study. Similar results were also detected in other bivalve molluscs. When green-lipped mussel Perna viridis exposed to 0.3 and 3 ug/L Bap for 18 days, SOD and GST capacity increased in the gills. However, CAT activity remained unchanged (Cheung et al., 2004). In scallop Chlamys farreri, SOD, CAT and GST enzymic activities in gills increased in short time at 0.5 and 1.0 ug/L concentration of Bap, but significantly depressed at 10.0 and 50.0 ug/L concentration (Pan et al., 2009). In mussels M. galloprovincialis, 10 ug/L Bap exposure for 7 days elevated the enzymic activities of CAT and GST enzymes in gills, but no change SOD activity (Maria and Bebianno, 2011). When clam Ruditapes philippinarum was exposed to 0.01 ug/L Bap for 21 days, the activities of SOD and GST were significant up-regulated at early stage of exposure (3 to 6 days), and then declined to the control level when the experiment terminated (Wang et al., 2011). After 6 days exposure of Bap at 56 ug/L, SOD activity in M. coruscus gills increased, but no significant induction of CAT activity (Chen et al., 2018). Perhaps the concentration and duration of Bap exposure, the sampling points, the sensitivity of various molluscs to Bap contribute to the different enzyme activities fluctuation. The MDA content exhibited a gradual increase with the exposure duration, suggesting that Bap exposure can time-dependently elevate the LPO level of blood clams, consequently leading to cytotoxicity. Similar results were also observed in scallop Chlamys farreri that MDA increased with the exposure time and there was a positive correlation between the MDA content and the concentration of Bap (Pan et al., 2009).
ROS generated from redox cycling progress of Bap cause macromolecules damage (Kim and Lee, 1997). The presence of 8-OHdG during DNA replication is known to mislead DNA templates at both modified and adjacent bases in vitro (Kuchino et al., 1987), and hence 8-OHdG is extensively studied as a biomarker for oxidative DNA damage. When mussels M. galloprovincialis was subjected to a wide dose-range of waterborne Bap, a significantly increased 8-OHdG level was detected in gills whereas no significant dose-response relationships were observed between 8-OHdG and Bap (Canova et al., 1998). However, when the same species was exposed to food-borne Bap, there was no apparent fluctuation in 8-OHdG in gills (Akcha et al., 2000). These researchers supposed that different exposure manners of Bap, viz. feed supply and waterborne, leaded to different 8-OHdG levels in the same mussel gills. In a field study to examine the efficiency and efficacy of biomarkers for environmental carcinogens monitoring, 8-OHdG levels in Perna viridis mussels between control and polluted sites showed no significant differences (Siu et al., 2008). In addition, over the complete 30-day exposure period, no significant correlations between 8-OHdG and organic contaminant concentrations in tissues were observed. However, at some sampling points in specific polluted sites, positive correlation was observed between Bap and 8-OHdG, whereas negative correlation was observed at another sampling point in another polluted site. Given these results, the researchers suggested that 8-OHdG was unlikely to be a good biomarker in field studies (Siu et al., 2008). The present results were likely to indicate a time-dependent increase of 8-OHdG in response to Bap exposure, that 8-OHdG levels in blood clams’ gills gradually increased and reached the peak value at 96 h in both 10 and 100 ug/L groups. This seemed at odds with previous researches, perhaps depending on the intensity and the duration of the stress applied on the one hand and on the susceptibility of the exposed living species on the other hand (Cossu et al., 2000). Notably, in in vitro study of the effects of metabolism enzymes on Bap-induced DNA damage in the scallop Chlamys farreri, 8-OHdG content increased significantly by inhibiting GST and SOD (Cai et al., 2016). Conversely, in the present study, the 8-OHdG content showed a significantly positive correlation with all examined antioxidants except SOD. The underlying mechanism for these discrepancies was uncertain. Despite of these, the present results demonstrated that Bap exposure could induce an oxidative DNA damage in blood clams’ gills, and this induction was time-dependent.
Neurotoxicity has been largely overlooked in the risk assessments of Bap (Chepelev et al., 2015). In a few studies using F344 rats as the model animals to investigate the neurotoxic effects for Bap, apparent neurotoxic symptoms such as physiological and autonomic activity dysfunction, weakened reaction to stimuli were exhibited after exposure to Bap (Saunders et al., 2006). ACh is a cholinergic neurotransmitter which plays a central role in regulation of the neuronal activity, generally used as an important index that reflects cholinergic nerve function (Li et al., 2012). However, ACh is very unstable and difficult to determine due to its rapid rate of hydrolysis, and therefore the functioning of the cholinergic system is usually observed through the activities of ChAT and AChE, which the former is responsible for ACh synthesis through catalyzed reaction of choline with acetoacetyl-CoA, whereas the latter exerts the hydrolysis of ACh into choline and acetic acid (Parsons et al. 1993). The measurement of AChE inhibition in marine organisms has been widely used as an indicator of neurotoxicity caused by environmental contamination. AChE activity has been demonstrated to be strongly inhibited by organophosphate and carbamate pesticides, and also by metals and surfactant agents (Guilhermino et al., 2000; Gill et al. 1991; Martinez-Tabche et al. 2001; Monserrat et al. 2002; Bainy et al.2006), causing dysfunction of the central nervous system. In mussels M. galloprovincialis, AChE activity was significantly reduced after exposure to Bap (Akcha et al., 2000; Banni et al., 2010). Similarly, in the present study, a remarkable decrease of AChE activity was also found in blood clams with exposure to Bap. The reduction of AChE activity could constrain the hydrolysis of the ACh (Shi et al., 2019), lead to neuro-transmission perturbation and cell apoptosis, and consequently weakening the neural system and causing the gill tissue in an obtuse state to external stimulus (Cong et al., 2017). These findings provided the direct evidence that Bap produce neurotoxicity in molluscs just as it has done in mammals, and AChE index could be used as an indicator to monitor Bap contamination. However, the underlying mechanism for inhibited AChE activity by Bap was unknown, perhaps irreversible or reversible binding to the catalytic site of the enzyme and potentiation of cholinergic effects provides certain contribution (Sarkar et al., 2006).
ChAT is the rate-limiting enzyme of ACh synthesis, which can be used as an indirect evaluation index of acetylcholine release level (Li et al., 2012). Even so, there were relatively few studies on ChAT as an indicator of neurotoxicity. In Alzheimer's disease patients, the activity of ChAT decreases 49–90% in the cerebral cortex, hippocampus and basal nucleus of telencephalon group, moreover, the degree of the decrease in activity is thought to be closely related to the degree of dementia (Lestaevel et al., 2011). In gills of mussels M. galloprovincialis caged for 60 days at Augusta, a site within the “Augusta-Melilli-Priolo” industrial area and thus suffering from severe petrochemical pollution, Maisano et al. (2017) reported an inhibition of both AChE and ChAT, indicating that cholinergic function was severely compromised, and this may result in impairment of the ciliary function and filtering activity of gills. Similarly, a statistically significant inhibition of AChE and ChAT in gills was found in mussels M. galloprovincialis collected from Lake Faro (Sicily, Italy), a natural confined brackish environment subjected to PAHs contamination (D’agata et al., 2014). Bap is a model PAHs, which is a major component of petrochemical pollutants, therefore these results indirectly suggested that ChAT in molluscs was also sensitive to Bap pollution. The current detection of significant inhibition of ChAT activity in the gills of Bap exposed blood clams further strengthens this view. Conversely, in gills of mussels caged for 30 days at Priolo, another site suffering as well as from petrochemical pollution, an inhibition of AChE coupled with an enhancement of ChAT was detected, suggesting that paracrine signalling mediated adaptive compensatory responses were possibly triggered to recover a regular physiological function of gills (Cappello et al., 2015).
DNA methylation has been demonstrated to be involved in the stress response to Bap exposure in mammals and model organisms. Smith and Hansch (2000) found that 5’-methylated cytosine at CpG islands was the preferred binding site for Bap in cigarette smoke, indicating that this site may be a target for lung cancer caused by Bap. In zebrafish Danio rerio, Bap exposure leaded to reduced global and gene specific DNA methylation (Fang et al., 2013; Corrales et al., 2014). Reduced DNA methylation of specific genes can increase the transcriptional expression level, which in turn involved in multiple pathogenic mechanisms including growth defects, embryo malformation and etc. Regrettably, so far, there have been no studies on the involvement of DNA methylation in response to Bap exposure in molluscs, and therefore no parallel data for us to compare the present result. However, the detected remarkably decrease of global DNA methylation here is consistent with previous findings in zebrafish, suggesting at least that DNA methylation in blood clams is involved in the stress response to Bap toxicity just as its correspondent epigenetic mechanism done in vertebrates. Notably, the majority of methylation in vertebrates occurs in non-coding intergenic regions of the DNA (Medvedeva et al., 2010). Hypomethylation of these regions, specifically gene promoters, typically results in prompting gene expression by opening transcriptional machinery (Kuramochi et al., 2001). Bap decreases the global DNA methylation level in zebrafish, leading to the induction of genes related to environmental stress by turning on the switch in gene promoters, and then participates in the fight against Bap toxicity. However, the mechanism maybe different in blood clams. In contrary to vertebrates, methylation of invertebrate DNA primarily exists in coding intragenic regions, suggesting a potentially different role for DNA methylation in invertebrates (Gavery and Roberts, 2010). The global DNA methylation reduction by Bap in blood clams might allow for fine-tuning of transcripts and of responses through transient methylation (Roberts and Gavery, 2012). Other mechanisms including exon skipping and transcription start sites substitution rather than controlling the switch in gene promoters may contribute to the variation in gene expression and resultly improve response to Bap toxicity (Roberts and Gavery, 2012). After exposure to copper, the global level of hydroxymethylcytosine in C. gigas, a most studied molluscan species in epigenetic scenario, was found to be reduced (Sussarellu et al., 2018). Similarly, a decrease on global DNA methylation level was also detected in Physa acuta with the exposure of vinclozolin and prednisolone, respectively (Sánchez-Argüello et al., 2016; Bal et al., 2017). On the contrary, cadmium exposure was founder to induces global cytosine (and possibly hydroxycytosine) hypermethylation in C. aspersus (Nica et al., 2017). These findings suggest that DNA methylation has potential to be associated with the response to multiple xenobiotics in molluscs, however the regulatory mechanism maybe different due to various organisms or pollutants.
The correlation analysis showed that global DNA methylation was significantly positively correlated with antioxidants activities. Given most previous reports have been demonstrated that DNA hypomethylation leads to the induction of genes related to environmental stress, we speculated that acute Bap exposure cause the reduction of global DNA methylation in gills of blood clams, which in turn trigger the antioxidants transcriptional expression, and consequently elevate the levels of antioxidants activities to confront Bap toxicity. Additionally, there was a positive correlation observed between global DNA methylation and choline enzymes activities, the underlying mechanism for this was uncertain. Considering that inhibition of AChE activity was associated with neurotoxicity, the current results at least suggested that Bap induced DNA hypomethylation indirectly correlated with neurotoxicity.
Herein, we performed a laboratory study to investigate the effects of Bap exposure on blood clam T. granosa. Multiple biomarkers, including histological changes, oxidative stress, neurotoxicity and global DNA methylation, were employed in the present study. Acute Bap exposure can induce significant morphological abnormalities in gills by generating oxidative stress and neurotoxicity. The global DNA methylation was inhibited, and possible correlated with elevated antioxidants activities against Bap toxicity. Meanwhile, Bap induced DNA hypomethylation may also be indirectly associated with neurotoxicity.