In the present study, microarray analysis was used to identify changes in gene expression underlying the pathological changes that occur when adult rats are coexposed to HD and x-ray. In our previous study, it was found that an 18-day priming exposure to the Sertoli cell toxicant HD resulted in a surprising attenuation of x-ray–induced germ cell apoptosis. Sensitization with HD exposure appears to induce an adaptive response, which alters the sensitivity of germ cells to x-rays. Using integrated bioinformatic techniques to analyze dose-response and co-exposure effects on gene expression, we identified individual genes and biological pathways underlying this attenuated germ cell response.
When studying mixed exposures, it is common practice to compare the gene expression profile after co-exposure to the gene expression profiles after exposure to the individual components of the mixture. With our study design involving multiple dose levels of each toxicant, this type of comparison could not be easily performed because of the complexity introduced by the dose-dependent effects of co-exposure. The phenotypic response of attenuated apoptosis following co-exposure was most unique; therefore, we focused primarily on the gene expression profile of the co-exposure group, dissecting the contribution of each toxicant to the co-exposure response.
HD pretreatment attenuated the downregulation by x-rays of IER3, ANGPTL4, CTGF, and ERRFI1 among other genes. IER3 is an immediate early response gene that has been shown to have an anti-apoptotic function in some cell types, promoting cell survival (Wu, 2003
). ANGPTL4 is an apoptosis survival factor for endothelial cells and also plays a role in lipid metabolism and insulin sensitivity (Kim et al., 2000
; Xu et al., 2005
). When secreted, CTGF stimulates proliferation of various cell types and extracellular matrix component production. CTGF can also be expressed as an intracellular protein in a cell cycle–dependent manner, causing cell cycle arrest (Kubota et al., 2000
). CTGF can interact with integrins on the Sertoli cell surface and may regulate essential Sertoli cell-germ cell interactions (O'Donnell et al., 2009
). ERRFI1 (ERBB receptor feedback inhibitor 1) is an immediate early response gene induced during periods of cell stress and can function as a tumor suppressor (Zhang and Vande Woude, 2007
). HD pretreatment also exerted a significant negative effect on many genes. In this group of genes, nearly all negative modifications by HD were an enhancement of x-ray–induced downregulation. Several of these genes (e.g., NASP, HSPB1, and ALKBH2) are involved in DNA replication, recombination and repair as well as cell death/apoptosis (e.g., GPI, CNTF, MAP3K11, COMP, and HSPB1). These changes are not surprising because x-ray exposure results in DNA damage that ultimately results in germ cell apoptosis. HD pretreatment also enhances the x-ray–induced downregulation of several genes whose products are involved in cellular growth and proliferation/cell cycle (e.g., SSRP1, NASP, GPI, PITPNM1, MAP3K11, and LOXL1) and also carbohydrate and lipid metabolism (PITPNM1, GHSR, CYGB, GPI, FBP1, ITPKA, and CNTF). Changes in carbohydrate and lipid metabolism have implications for reduced Sertoli cell support of and communication with germ cells.
Some other genes that are strongly further downregulated by HD co-exposure are LOXL1, GHSR, and PRSS21 (protease, serine, 21). GHSR and LOXL1 exhibit some of the greatest negative HD effects and recent studies have begun to reveal a role for the products of these genes in the male reproductive system. LOXL1−/− male mice exhibit decreased fertility and decreased sperm production, although the mechanisms behind these effects are unknown (Wood et al., 2009
). Ghrelin is the endogenous ligand for GHSR. Locally expressed ghrelin in the testis inhibits hCG- and cAMP-stimulated testosterone secretion in vitro
and also causes a reduction in steroidogenic gene expression (Lorenzi et al., 2009
). PRSS21, also called TEST1 or TESTISIN, is a serine protease expressed by premeiotic primary spermatocytes in the cytoplasm and on the plasma membrane and is suggested to be involved in germ cell maturation (Hooper et al., 1999
). Whereas the exact role of TEST1 is not known, it may participate in proteolytic events necessary for germ cell migration, or in the exchange of soluble factors or cell surface interactions between germ cells and Sertoli cells (Hooper et al., 1999
In addition to focusing on individual genes altered by HD on top of x-ray, pathways analysis was used to gain a better understanding of the molecular changes underlying the co-exposure response. Biological pathways with the greatest modification of gene expression by HD exposure were identified, and within these pathways several genes of interest were revealed. A significant influence of HD on genes involved in cell cycle was discovered. Looking at the genes within these pathways below the threshold of p
< 0.05, most are negatively affected by HD, suggesting that there may be cell cycle arrest. HD-mediated attenuation of genes that promote cell proliferation may cause a reduction in germ cell proliferation, rendering them more resistant to x-ray exposure. HD-exposed rats exhibited increased duration of the spermatogenic cycle that was attributed to reduced progression of 5-bromodeoxyuridine (BrdU)-labeled cells (Rosiepen et al., 1995
). Compromised Sertoli cell-mediated transport of germ cells may be the cause for decreased BrdU labeling during specific stages. However, impaired division of spermatogonia may also explain the reduction in BrdU-labeled germ cells. Among the other genes within these cell cycle–related gene sets, there is an HD-mediated enhancement of CDKN1A (cyclin-dependent kinase inhibitor 1A, p21), a cell cycle inhibitor that is controlled by p53. p53-dependent cell cycle arrest in response to cell stress is mediated by CDKN1A/p21, and this growth arrest mediated by p21 can inhibit apoptosis (Yu and Zhang, 2005
). Alternatively, there is decreased expression of several cyclins (CCNA1, CCNB1, CCNE1, CCNG1, and CCND2) and other genes involved in the promotion of cell cycle progression following co-exposure. For example, NASP (nuclear autoantigenic sperm protein [histone binding]), which is decreased by HD, is regulated by the cell cycle and functions in the transport of histones to the nucleus of dividing cells (Richardson et al., 2000
The attenuation of germ cell apoptosis was the most remarkable histopathologic observation following co-exposure to HD and x-ray, therefore it was not surprising that the cell death/apoptosis pathway was overrepresented among significant genes. Examining the genes within this pathway, as well as considering those genes significantly altered by x-ray exposure (Supplementary table 1
), there is a strong trend toward attenuation of x-ray–induced proapoptotic genes by HD co-exposure. CCNG1 (cyclin G1), BBC3 (Bcl2-binding component 3), and AEN (apoptosis-enhancing nuclease) are all proapoptotic genes that were significantly increased by x-ray exposure with HD-mediated attenuation following co-exposure. HD co-exposure prevents the induction of CCNG1 caused by x-ray. CCNG1 is a transcriptional target of p53 that regulates p53 and its functions, including apoptosis (Kimura and Nojima, 2002
). Under certain cellular conditions, CCNG1 can also negatively regulate p53 and promote cell growth rather than cell cycle arrest (Chen, 2002
). As discussed earlier, a decrease in germ cell proliferation, which could be related to attenuated CCNG1 upregulation, may protect germ cells from x-ray. Attenuation of x-ray–induced BBC3 and AEN was also detected by gene array analysis. BBC3, also known as PUMA (p53-upregulated modulator of apoptosis), mediates p53-induced apoptosis by either preventing the action of the antiapoptotic proteins Bcl-2 and Bcl-XL
, or by promoting p53 dissociation from Bcl-XL
, allowing p53 to activate Bax (Wang et al., 2007
). AEN enhances apoptosis following ionizing radiation through its DNase activity (Lee et al., 2005
). BBC3/PUMA and AEN are increased by 1.7- and 3.2-fold, respectively, after exposure to 5 Gy x-ray, with their expression attenuated to control levels following co-exposure. These notable differences in induction between x-ray alone and co-exposure, along with the documented roles of BBC3/PUMA and AEN in the apoptotic pathways that are activated following radiation exposure, make these genes strong candidates for further investigation. In addition to these modifications of proapoptotic genes, there was HD-dependent enhancement of antiapoptotic genes. NFKBIA (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha), better known as IκBα, functions to inhibit nuclear factor κB (NF-κB) proteins by sequestering them in the cytoplasm in an inactive state (Jacobs and Harrison, 1998
) and is enhanced by co-exposure to HD. This enhancement would be expected to reduce apoptosis as NF-κB activation is proapoptotic in the irradiated testis (Rasoulpour and Boekelheide, 2007
In conclusion, we have derived new insights into the mechanisms underlying the phenotypic effects of HD and x-ray co-exposure by investigating global gene expression in the testis, focusing on the influence of HD on x-ray–induced gene alterations. The less supportive environment induced by HD pretreatment results in adaptation of the germ cells and an increased resistance to subsequent insults by x-ray exposure. The modulation of gene expression caused by HD sensitization corresponds well with the phenotypic characteristics resulting from HD pretreatment. Particularly, the HD-dependent attenuation of several proapoptotic genes correlates with the attenuated germ cell apoptosis. Additional studies are underway to investigate these alterations in apoptotic genes, specifically BBC3/PUMA and AEN. In the current study, gene expression analysis was performed using RNA isolated from whole testis; however, the attenuated apoptosis is stage specific (Yamasaki et al., 2010
). More focused studies investigating the expression of these apoptotic genes in specific stages by qRT-PCR, which is more sensitive than microarray analysis, will provide further insight into the current findings. In addition to these alterations in apoptotic genes, HD modulated several signaling pathways of interest, including cell cycle/cell division process and cell cycle/G1/S phase transition that may mechanistically explain this reduced apoptotic response. Abnormal Sertoli cell support of germ cells and altered communication between Sertoli cells and germ cells by HD pretreatment likely contribute to the attenuation of germ cell apoptosis. This may be due to a role of Sertoli cells in regulating germ cell apoptosis following DNA damage. Alternatively, reduced Sertoli cell support may result in decreased germ cell division and therefore, reduced vulnerability to x-rays that target actively dividing cells. Future studies will explore further this possible disruption of germ cell division by HD exposure.