Induction of NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity

Abstract

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7-and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0-and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIELs-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure

“…Furthermore, we have also shown that NEILs, which are distinct from OGG1 in structural features and reaction mechanisms, and which act on different substrates (Dou et al, 2003; Englander and Ma, 2006, Ma et al, 2011), are also involved in the repair of aniline-induced DNA damage in the spleen of aniline-treated rats. Although the role of NTH1 and APE1 in DNA repair and transcriptional regulation has been widely investigated, the roles of NTH1 and APE1 in the regulation of aniline-induced splenotoxic response have not been evaluated.…”

“…Earlier studies from our laboratory have shown that aniline exposure results in iron overload and oxidative stress in the spleen (Khan et al, 1997, 1999, 2003a, 2003b). More importantly, our studies have shown increased oxidative DNA damage in the spleen of rats following aniline exposure (Wu et al, 2005; Ma et al, 2008, 2011), which could potentially lead to mutagenic and/or carcinogenic responses.…”

“…One group of animals was given 0.5 mmol/kg/day of aniline (~97%; Sigma-Aldrich, Milwaukee, WI) via drinking water (pH of the solution adjusted to ~6.8) (Khan et al, 1993, 1999; Ma et al, 2008, 2011), whereas the other group received drinking water only and served as controls. The choice of aniline dose was based on our earlier studies that showed significant increases in lipid peroxidation, protein oxidation and oxidative DNA damage (oxidative stress) in the spleen (Khan et al, 1999; Ma et al, 2008, 2011). After 30 days, the rats were euthanized under nembutal (sodium pentobarbital) anesthesia and the spleens were removed immediately, blotted, weighed and stored at −80 °C until further analysis.…”

“…In mammalian cells, there are several different DNA glycosylases with overlapping substrate specificities that remove oxidative DNA base lesions. These glycosylases include 8-oxoguanine glycosylase 1 (OGG1), endonuclease III homologue 1 (NTH1), and the Nei-like DNA glycosylases (NEIL1/2) (Rolseth et al, 2008; Mori et al, 2009; Ma et al, 2011). Excision of DNA damaged bases by OGG1 and NTH1 glycosylases is followed by a common pathway involving an AP-endonuclease (APE) to generate 3’ OH terminus at the damage site.…”

Regulation of DNA glycosylases and their role in limiting disease

“…In addition, transcriptome analyses of tissue from rats fed quercetin showed an induction of Mutyh expression [48,81]. Levels of Neil1 mRNA have been shown to be elevated following ROS [43] and aniline treatments [143]. …”