The development of massive sequencing has provided a relatively inexpensive method to obtain the transcriptome of a species. Taking advantage of this technique, we used a previously obtained transcriptome of P. acuta to identify 18 genes related to different pathways of interest in ecotoxicology and then examined how exposure to phthalates changed the transcription of these genes. The processes of interest include DNA repair, the stress response, detoxification, apoptosis, immunity, energy reserves, and lipid transportation. There is a growing interest in combining ecologically relevant endpoints with biochemical and molecular parameters to seek a more integrative analysis. In this sense, increasing the number of described genes will allow for the design of standard arrays that could be used in combination with toxicity tests. In this way, initiatives such as the Adverse Outcome Pathway wiki24 will increase its relevance in assessing old and new compounds and provide putative mechanisms of action to explain the differences to the animals’ specific physiology. Furthermore, increasing knowledge at the molecular level in P. acuta supports its use as a representative of freshwater gastropods in toxicity analysis. There is a lack of model freshwater mollusks, which is one of the animal groups whose pollution response is currently less known.
The 18 newly identified genes evaluated in this work show homology with those previously described in other species, as expected, mainly with the freshwater snail Biomphalaria glabrata, which belongs to the Planorbidae family. rad21 and rad50 are both involved in DNA repair: rad21 is an essential gene encoding a DNA double-strand break repair protein21, and rad50 is a member of the protein complex MRN (including Mre11, RAD50, and Nbs1) that functions in DNA double-strand break repair to recognize and process DNA ends as well as a signal for cell cycle arrest25. There is very little information about these genes in mollusks, with only one report in Crassostrea gigas for rad5026. The relevance of these genes is that their detection can be combined with other methodologies, such as the comet assay, to perform an integrated study to determine whether a compound is genotoxic and whether the organism has the ability to compensate for the damage.
The Cat and SOD Mn genes allow us to evaluate the status of oxidative stress. Oxidative stress analysis is usually focused on biochemical parameters, such as enzyme activity. However, it should also include a transcriptional activity study because it can provide additional information about the mid- and long-term responses. Protein turnover can also be relevant in the response, especially in chronic exposure to toxicants. Detoxification mechanisms are also important to assess the response to toxicants. GST activity is one of the most used methods to assess detoxification27, but it does not differentiate between the members involved. The situation is similar regarding cytochrome P450s, which show high diversity with many roles in the cell28. Our identification of the Cyp72a15 gene increases the number of cytochromes 450 s described in P. acuta. Evaluating changes in these genes can help to elucidate how the organism can process the toxicants.
The sHSP17.9 and HSC70-4 genes extend the battery of genes available to assess the stress response of P. acuta. sHSP17.9 is difficult to match with other species’ genes because while they all have an alpha-crystallin domain, there is no other sequence that presently allows for homology to be established. Additional functional studies will help to search for homology. It is worth mentioning that HIF1α offers a new aspect of stress related to hypoxia29. The stress response mainly focuses on the canonical heat shock proteins, so other mechanisms involved in specific stresses, such as hypoxia, are usually neglected. With the identification of HIF1a in P. acuta, researchers can evaluate the effect of a toxicant on oxygen intake in this species.
The remaining identified genes allow for the analysis of pathways that can also be altered by toxicants, like apoptosis (AIF3), the immune system (ApA), energy reserves (PYGL), and lipid transport (ORP8). To our knowledge, in this study these genes have been analyzed for the first time concerning pollution in freshwater mollusks. The last three genes, DNMT1, KATB6, and HDAC1, are involved in epigenetic mechanisms. There is increasing evidence that epigenetic regulation is one of the long-term effects of toxicants. However, the genes involved in this process in invertebrates are still poorly represented in toxicity analysis. The description of these three genes opens the possibility of analyzing their role in the epigenetic response and its relevance in the transgenerational effects that have started to be described with different toxicants30,31,32.
Plastics in the environment are a growing problem. During the degradation process, the polymers themselves and the compounds used as additives, including phthalates, are released. Hence, the presence of phthalates is increasing in the environment5,33,34. We analyzed three phthalates in this work, namely BBP, DEP, and DEHP; they showed a differential impact in P. acuta. DEP and DEHP, did not alter any of the mRNA levels. Researchers have described previously that both phthalates can alter the physiology of invertebrates16,35,36,37,38, including mollusks39,40,41. Other phthalates can also alter development and growth, which could be related to the endocrine-disrupting activity described for those chemicals. The molecular mechanisms involved are still under investigation, but some data are available. In the clam Venerupis philippinarum, DEHP alters the immune response40. In H. diversicolor, DBP affects oxidative stress, lipid and energy metabolism, and osmoregulation17. In other invertebrates, including Chironomus riparius42, Drosophila melanogaster43, and Caenorhabditis elegans15, phthalates alter endocrine pathways. The changes affect the ecdysone response as well as the expression of insulin-like peptide. Other pathways are also affected by phthalates, such as oxidative stress and detoxification routes44 and the stress response14. Finally, in C. elegans, exposure to environmentally relevant concentrations of diethylhexyl phthalate produces genomic instability by altering the expression of genes involved in DNA repair during meiosis37. It is clear then that phthalates can have a broad spectrum of actions in the cell, with a significant alteration of metabolism but primarily affecting oxidative stress and the endocrine system.
The previous studies performed in mollusks have revealed alterations in several physiological processes; the analyzed molecular mechanisms mainly involved oxidative stress and immunity17,41. A recent review of the impact of phthalates on aquatic animals summarizes the effects observed, suggesting that activation of the detoxification system (cytochrome P450s) and endocrine system receptors of aquatic animals cause oxidative stress, metabolic disorders, endocrine disorders, and immunosuppression8. It would activate a cascade response that could cause genotoxicity and cell apoptosis, resulting in the disruption of growth and development. Considering this, the absence of a response observed in P. acuta exposed to DEP and DEHP is striking. The differences observed can be assigned to the type of analysis (molecular vs. physiological), the exposure time (1 week vs. a few hours or days), the concentration used (μg/L vs. mg/L), and evidently, the species used. Additional research will help elucidate the differential response in P. acuta compared with other organisms. However, it is essential to highlight that the obtained results suggest that P. acuta can manage the environmentally relevant doses of DEP and DEHP used in this work. This species may be less sensitive to these phthalates, but this eventually will require further research, including the use of other methodological approaches, to confirm it.
In contrast to DEP and DEHP, BBP showed a marked effect: it increased the mRNA levels of almost all the analyzed genes. It is essential to consider that most studies on invertebrates that involve transcriptional activity analysis use arthropods and short exposure times14,44,45,46. Limited data are available on mollusks and, usually, they are marine representatives40,47. To our knowledge, this is the first study on a freshwater snail that shows that BBP can produce a substantial effect on cell metabolism. Several of the altered pathways can explain, in some way, the effects observed in other organisms, like DNA repair by the alteration of rad21 and rad50, which are related to DNA damage, or the alteration of the genes involved in histone and DNA modification (KAT6B, HDAC1, and DNMT1), which are related to epigenetic regulation. Apoptosis, which phthalates can also alter, also seems to be modulated in P. acuta by altering the AIF3 and the casp3 genes. Furthermore, the three phases of the detoxification could be acting since the genes tested (three cytochrome P450s, three GSTs, and MRP-1) were upregulated.
Genes involved in oxidative stress and the stress response were also altered, as shown by the changes in the mRNA levels of Cat, SODs, stress proteins, and the hypoxia-related transcription factor genes. These changes support the alteration of oxidative stress, the stress response, and detoxification, backing previous analysis and adding new insight about the mechanisms involved in modulating these processes. In this sense, the absence of changes in GSTm1 supports a differential role for each GST family member in the response to toxicants. The altered acetylcholinesterase mRNA level also suggests effects in the nervous system, requiring additional research to elucidate the damage to the central nervous system. Finally, the alteration of PYGL, ApA, and ORP8, involved in energy metabolism, immunity, and lipid transport, respectively, shows that P. acuta responds to BBP in a way that has been observed in other organisms. In summary, the present gene profile obtained in response to BBP in P. acuta supports the proposed mechanisms and cellular processes in studies with other animals8. Immunity, oxidative stress, the stress response, detoxification, apoptosis, epigenetic modulation, DNA repair, lipid metabolism, and energy metabolism are modulated. The nervous system could also be affected. Of note, some genes showed differences in transcription based on the phthalate concentration. These findings suggest there are subtle differences, and additional kinetic analysis is required to elucidate early and late activated genes and the relevance of the damage for the population’s future.
The obtained results are in line with previous studies in other organisms, which have confirmed that BBP can induce different types of damage such as apoptosis48, genotoxicity49, oxidative stress50, stress response activation45, or endocrine disruption14. Although there are studies in invertebrates showing the impact on development and other physiological processes39,51, most of them did not focus on the putative mode of action, with only a few of them trying to delve into the response mechanisms. Here we have shown that BBP can extensively affect the cell transcriptional activity in P. acuta. These results could be considered to reflect specific alterations on these pathways. This scenario would mean that BBP is the most active phthalate in P. acuta, with a broad spectrum of action and a potential effect on many pathways. However, the more probable picture is something that has been recently proposed: alterations in the oxidative stress response and the endocrine system cause a cascade of responses that affect different pathways and ultimately block growth and development8. It is relevant to keep in mind that BBP is a known endocrine disruptor47. A recent study in Daphnia magna provides some insight. Specifically, RNA-Seq revealed that genes involved in signal transduction, cell communication, and embryonic development were significantly down-regulated, while those related to biosynthesis, metabolism, cell homeostasis, and redox homeostasis were remarkably upregulated upon BBP exposure46. Although the organism and the stage analyzed are different from our study, those results support the idea that BBP can simultaneously alter multiple pathways, and it fits better with the regulatory role of the endocrine system and the extensive affection by oxidative stress.
As stated before, the results obtained in this work show that DEP and DEHP had no apparent effect to P. acuta after 1 week exposure to environmentally relevant concentrations. However, BBP showed a strong effect. The difference in response could be due to several reasons that need to be explored in future work. One possibility is the structure of each compound. In this sense, BBP has two benzene rings while DEP and DEHP have only one. This factor could determine the biological activity of these compounds. Another possibility is that DEP and DEHP have effects earlier than the time studied, and the cell returned to the basal state, being able to process and remove the compounds. Finally, it cannot be dismissed that DEP and DEHP are not toxic to P. acuta, at least at environmentally relevant concentrations. In any case, BBP alters the metabolism of this species and produces a broad impact on different pathways. Additional research should be done in P. acuta and other freshwater species to determine the impact on organisms based on the freshwater ecosystem food web.
Source: Ecology - nature.com
