The cellular and subcellular targeting of the individual toxicants in a mixture determine the toxicological outcome. The current study has demonstrated that CBZ and HD interact in an agonistic way at both the phenotypic level and at the gene level. Combined exposure to HD and CBZ has synergistic effects on the expression of specific genes, including Clca2/Clca4l and Loxl1, whose altered expression may underlie the enhanced testicular toxicity. Clca2/Clca4l and Loxl1 were identified as candidate genes involved in the enhanced co-exposure response through microarray analysis. Similar to a previous study [7
], microarray data were summarized across different dose levels and treatment groups (toxicant combinations) using a statistical method that estimates the extent to which HD modifies gene expression above and beyond the CBZ–induced gene expression for each gene during co-exposure. This allowed for the identification of genes that are more altered in expression by co-exposure as compared to CBZ alone. These genes are likely to be related to the enhanced pathology. All 5 genes that were significantly altered by HD on top of CBZ exhibited agonistic, or synergistic, gene expression alterations with co-exposure as detected by gene array analysis, which mirrors the synergistic effects of co-exposure on testicular toxicity. Although previous studies have demonstrated that microarray analysis using RNA isolated from whole testis tissue may not provide the most reliable information for hypothesis generation [10
], both Clca2 and Loxl1 proved to be promising genes of interest likely to play a role in the enhanced co-exposure toxicity.
Although most of the microarray expression alterations were confirmed by qRT-PCR, there were some discrepancies. Some of these differences may be due to the differences in sensitivity and the different dynamic ranges of the two different technologies. It is also important to remember that the estimated “effects” used to identify candidate genes for additional analysis were derived from the microarray expression data for all 9 different treatment groups across different dose levels, while the qRT-PCR was performed only on the high doses. As described in the results section, the microarray fold-changes for those specific treatment groups are quite similar to the qRT-PCR-detected gene alterations in those same treatment groups. The only real discrepancy between the 3 hr qRT-PCR results and the microarray results (also obtained at the 3 hr time point) is the significant increase in Macrod1 detected by qPCR after 1% HD exposure. No significant changes were detected by array for this treatment group. All other significant changes detected by qRT-PCR confirm the microarray results. The expression pattern of Tubb3 (tubulin, beta 3), as measured by qRT-PCR, provides confidence in these data. Tubulin synthesis is controlled by autoregulation, whereby non-polymerized, free tubulin monomers provide feedback, regulating mRNA levels of tubulin [11
]. Since CBZ and HD inhibit or promote microtubule assembly, respectively, one would expect CBZ to decrease tubulin mRNA levels and HD to increase tubulin mRNA levels. As would be expected, an increase in tubulin mRNA levels after HD exposure is observed in the current study, with only a slight decrease detected after CBZ exposure. These tubulin expression results provide confidence in the quality of the RNA used for microarray analysis and the resulting data.
Loxl1 catalyzes the oxidation of lysine residues of collagen fibrils and elastins in the extracellular matrix, which controls the cross linking and deposition of elastins. Loxl1 is localized specifically to sites of elastogenesis and plays a role in elastin homeostasis [12
]. Interestingly, Loxl1 −/− male mice exhibit lower sperm production and reduced fertility, with no apparent histologic differences [13
]. Loxl1 appears to play a role in male sexual development and fertility, but the specific mechanism(s) are currently unknown. The substantial reduction in Loxl1 mRNA in the present study suggests that this gene product may also play a role in the toxicological response of the testis. A closely related family member to Loxl1, lysyl oxidase (LO), is able to prevent the activation of NFκB by inhibiting the signaling pathways that lead to its activation [14
], so one would anticipate that reduced LO or Loxl1 levels would lead to greater activation of NFκB. Given that NFκB is pro-apoptotic in the testis [15
], the reduction in Loxl1 observed in the current study corresponds with, and may mechanistically explain, the enhanced injury and apoptosis that occurs following HD and CBZ co-exposure. One might also speculate that altered Loxl1 expression during injury may impact germ cell support through alterations in cell-matrix interactions and cell junction dynamics related to elastic fiber homeostasis. Further mechanistic studies are required to better understand the role of Loxl1 in the testis, both under normal conditions and following toxicant exposure.
In addition to the detected changes in Loxl1 expression after co-exposure, altered expression of the chloride channel Clca2/Clca4l in the testis likely has significant consequences, although the specific role and localization of this particular chloride channel in the testis has not yet been investigated. Chloride channels have been demonstrated to play a role in regulating the volume of spermatozoa, the regulation of seminiferous tubule fluid formation, and in controlling the ionic environment in the testis [16
]. The survival of germ cells and the proliferation of spermatogonia is dependent on the production of an acidic microenvironment. Lactate secretion by Sertoli cells helps to maintain this acidic microenvironment and the chloride currents in Sertoli cells may be involved in the proton-linked lactate production in Sertoli cells [18
]. Other members of the Clca protein family appear to play a role as cell-cell adhesion molecules, which may be important in the testis [20
]. The critical role of chloride channels in the testis is observed in the severe degeneration in mouse testes that occurs when chloride channel expression is disrupted [19
While Clca2/Clca4l is a strong candidate for further analysis, Clca2/Clca4l staining was not performed because there are no commercially available antibodies to detect the rat protein. The quantification of Loxl1 immunostaining reflects the gene expression as measured by qRT-PCR at 3 h, however the changes in protein levels were not large in magnitude or statistically significant. This non-significance may be attributed to the 24 h time point at which protein expression was examined. The greatest change in Clca2/Clca4l gene expression occurs at 24 h, with the magnitude of change for Loxl1 being consistent between 3 and 24 h. This may reflect a sustained alteration in Loxl1 expression over these time points, or may indicate a peak in gene expression alteration between these 2 time points. This may indicate that significant alterations in Loxl1 protein levels would follow at a later time point.
The localization of Loxl1 within the testis has not been previously investigated. Loxl1 was found to be localized to several areas within the seminiferous tubules, including immature spermatozoa tails, the cytoplasm of Sertoli cells, the basement membrane of the seminiferous tubule, blood vessels, and acrosomes. The most intense staining was localized to the acrosomes. Interestingly, the localization of Loxl1 to the acrosome is similar to the localization of clusterin, a glycoprotein that is produced constitutively by Sertoli cells and has been well-studied in the testis [21
]. Clusterin has been found to localize to the cytoplasm of Sertoli cells, in the heads and tails of late spermatids and released spermatozoa, and at the acrosome [21
]. Recently, Loxl1 and clusterin have been identified as major genetic variants in pseudoexfoliation syndrome, which have been shown to co-localize in ocular tissues of patients with this disease [23
]. There is a possibility that these proteins co-localize in the testis as well, given the similar areas of localization. Further research will need to be performed to confirm this and also to determine the potential relationship of these proteins in the testis. The potential interactions of Loxl1 and clusterin in the testis at the protein level may also extend to the functional level, as clusterin has been implicated in protecting surviving cells after damage.
In summary, these studies have revealed candidate genes underlying the synergistic disruption of spermatogenesis that occurs following HD and CBZ co-exposure. CBZ and HD interact in an agonistic way at the gene level, reflective of the agonistic effects on testicular toxicity. Loxl1 and Clca2/Clca4l are both reduced in expression following co-exposure and appear to play critical roles in the testis. Further investigation is needed to determine the specific roles of Loxl1 and Clca2/Clca4l in the testicular response to damage. The results of the current study have also demonstrated, for the first time, the localization of Loxl1 in the testis. Additional mechanistic studies will reveal the functional significance of this localization in the testis as well as the testicular role of Loxl1, both under normal conditions and following toxicant exposure.
- CBZ and HD interact in a synergistic way at both the histological level and at the gene level
- Combined exposure to HD and CBZ has synergistic effects on the expression of specific genes, including Clca2/Clca4l and Loxl1.
- The localization of Loxl1 within the testis has not been previously investigated and Loxl1 was found to be localized to several areas within the seminiferous tubules with the most intense staining localized to the acrosomes.