Arginine methylation of caspase-8 controls life/death decisions in extrinsic apoptotic networks.

Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.

Modulation of extrinsic apoptotic pathway by intracellular glycosylation.

The importance of post-translational modifications (PTMs), particularly O-GlcNAcylation, of cytoplasmic proteins in apoptosis has been neglected for quite a while. Modification of cytoplasmic proteins by a single N-acetylglucosamine sugar is a dynamic and reversible PTM exhibiting properties more like phosphorylation than classical O- and N-linked glycosylation. Due to the sparse information existing, we have only limited understanding of how GlcNAcylation affects cell death. Deciphering the role of GlcNAcylation in cell fate may provide further understanding of cell fate decisions. This review focus on the modulation of extrinsic apoptotic pathway via GlcNAcylation carried out by O-GlcNAc transferase (OGT) or by other bacterial effector proteins.

RL2 Enhances the Elimination of Breast Cancer Cells by Doxorubicin.

RL2 (recombinant lactaptin 2), a recombinant analogon of the human milk protein Κ-Casein, induces mitophagy and cell death in breast carcinoma cells. Furthermore, RL2 was shown to enhance extrinsic apoptosis upon long-term treatment while inhibiting it upon short-term stimulation. However, the effects of RL2 on the action of chemotherapeutic drugs that induce the intrinsic apoptotic pathway have not been investigated to date. Here, we examined the effects of RL2 on the doxorubicin (DXR)-induced cell death in breast cancer cells with three different backgrounds. In particular, we used BT549 and MDA-MB-231 triple-negative breast cancer (TNBC) cells, T47D estrogen receptor alpha (ERα) positive cells, and SKBR3 human epidermal growth factor receptor 2 (HER2) positive cells. BT549, MDA-MB-231, and T47D cells showed a severe loss of cell viability upon RL2 treatment, accompanied by the induction of mitophagy. Furthermore, BT549, MDA-MB-231, and T47D cells could be sensitized towards DXR treatment with RL2, as evidenced by loss of cell viability. In contrast, SKBR3 cells showed almost no RL2-induced loss of cell viability when treated with RL2 alone, and RL2 did not sensitize SKBR3 cells towards DXR-mediated loss of cell viability. Bioinformatic analysis of gene expression showed an enrichment of genes controlling metabolism in SKBR3 cells compared to the other cell lines. This suggests that the metabolic status of the cells is important for their sensitivity to RL2. Taken together, we have shown that RL2 can enhance the intrinsic apoptotic pathway in TNBC and ERα-positive breast cancer cells, paving the way for the development of novel therapeutic strategies.

The cross-talk of autophagy and apoptosis in breast carcinoma: implications for novel therapies?

Breast cancer is still the most common cancer in women worldwide. Resistance to drugs and recurrence of the disease are two leading causes of failure in treatment. For a more efficient treatment of patients, the development of novel therapeutic regimes is needed. Recent studies indicate that modulation of autophagy in concert with apoptosis induction may provide a promising novel strategy in breast cancer treatment. Apoptosis and autophagy are two tightly regulated distinct cellular processes. To maintain tissue homeostasis abnormal cells are disposed largely by means of apoptosis. Autophagy, however, contributes to tissue homeostasis and cell fitness by scavenging of damaged organelles, lipids, proteins, and DNA. Defects in autophagy promote tumorigenesis, whereas upon tumor formation rapidly proliferating cancer cells may rely on autophagy to survive. Given that evasion of apoptosis is one of the characteristic hallmarks of cancer cells, inhibiting autophagy and promoting apoptosis can negatively influence cancer cell survival and increase cell death. Hence, combination of antiautophagic agents with the enhancement of apoptosis may restore apoptosis and provide a therapeutic advantage against breast cancer. In this review, we discuss the cross-talk of autophagy and apoptosis and the diverse facets of autophagy in breast cancer cells leading to novel models for more effective therapeutic strategies.

Regulation of extrinsic apoptotic signaling by c-FLIP: towards targeting cancer networks.

The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP). In addition to its key role in apoptosis, c-FLIP may exert other cellular functions, including control of necroptosis, pyroptosis, nuclear factor κB (NF-κB) activation, and tumorigenesis. To gain further insight into the molecular mechanisms of c-FLIP action in cancer networks, we focus on the structure, isoforms, interactions, and post-translational modifications of c-FLIP. We also discuss various avenues to target c-FLIP in cancer cells for therapeutic benefit.

Impact of human CD95 mutations on cell death and autoimmunity: a model.

CD95/Fas/APO-1 can trigger apoptotic as well as nonapoptotic pathways in immune cells. CD95 signaling in humans can be inhibited by several mechanisms, including mutations in the gene encoding CD95. CD95 mutations lead to autoimmune disorders, such as autoimmune lymphoproliferative syndrome (ALPS). Gaining further insight into the reported mutations of CD95 and resulting alterations of its signaling networks may provide further understanding of their presumed role in certain autoimmune diseases. For illustrative purposes and to better understand the potential outcomes of CD95 mutations, here we assign their positions to the recently determined 3D structures of human CD95. Based on this, we make certain predictions and speculate on the putative role of CD95 mutation defects in CD95-mediated signaling for certain autoimmune diseases.

The role of death domain proteins in host response upon SARS-CoV-2 infection: modulation of programmed cell death and translational applications.

The current pandemic of novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) poses a significant global public health threat. While urgent regulatory measures in control of the rapid spread of this virus are essential, scientists around the world have quickly engaged in this battle by studying the molecular mechanisms and searching for effective therapeutic strategies against this deadly disease. At present, the exact mechanisms of programmed cell death upon SARS-CoV-2 infection remain to be elucidated, though there is increasing evidence suggesting that cell death pathways play a key role in SARS-CoV-2 infection. There are several types of programmed cell death, including apoptosis, pyroptosis, and necroptosis. These distinct programs are largely controlled by the proteins of the death domain (DD) superfamily, which play an important role in viral pathogenesis and host antiviral response. Many viruses have acquired the capability to subvert the program of cell death and evade the host immune response, mainly by virally encoded gene products that control cell signaling networks. In this mini-review, we will focus on SARS-CoV-2, and discuss the implication of restraining the DD-mediated signaling network to potentially suppress viral replication and reduce tissue damage.

The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells.

Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2-TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.

Controlling Cell Death through Post-translational Modifications of DED Proteins.

Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.

Interplay Between Mitophagy and Apoptosis Defines a Cell Fate Upon Co-treatment of Breast Cancer Cells With a Recombinant Fragment of Human κ-Casein and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

A recombinant fragment of human κ-Casein, termed RL2, induces cell death of breast cancer cells; however, molecular mechanisms of RL2-mediated cell death have remained largely unknown. In the current study, we have decoded the molecular mechanism of the RL2-mediated cell death and found that RL2 acts via the induction of mitophagy. This was monitored by the loss of adenosine triphosphate production, LC3B-II generation, and upregulation of BNIP3 and BNIP3L/NIX, as well as phosphatase and tensin homolog-induced kinase 1. Moreover, we have analyzed the cross talk of this pathway with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon combinatorial treatment with RL2 and TRAIL. Strikingly, we found two opposite effects of this co-treatment. RL2 had inhibitory effects on TRAIL-induced cell death upon short-term co-stimulation. In particular, RL2 treatment blocked TRAIL-mediated caspase activation, cell viability loss, and apoptosis, which was mediated via the downregulation of the core proapoptotic regulators. Contrary to short-term co-treatment, upon long-term co-stimulation, RL2 sensitized the cells toward TRAIL-induced cell death; the latter observation provides the basis for the development of therapeutic approaches in breast cancer cells. Collectively, our findings have important implications for cancer therapy and reveal the molecular switches of the cross talk between RL2-induced mitophagy and TRAIL-mediated apoptosis.

Modulation of CD95-mediated signaling by post-translational modifications: towards understanding CD95 signaling networks.

CD95 is a member of the death receptor family and is well-known to promote apoptosis. However, accumulating evidence indicates that in some context CD95 has not only the potential to induce apoptosis but also can trigger non-apoptotic signal leading to cell survival, proliferation, cancer growth and metastasis. Despite extensive investigations focused on alterations in the expression level of CD95 and associated signal molecules, very few studies, however, have investigated the effects of post-translational modifications such as glycosylation, phosphorylation, palmitoylation, nitrosylation and glutathionylation on CD95 function. Post-translational modifications of CD95 in mammalian systems are likely to play a more prominent role than anticipated in CD95 induced cell death. In this review we will focus on the alterations in CD95-mediated signaling caused by post-translational modifications of CD95.

Decoding the sweet regulation of apoptosis: the role of glycosylation and galectins in apoptotic signaling pathways.

Glycosylation and glycan-binding proteins such as galectins play an important role in the control of cell death signaling. Strikingly, very little attention has been given so far to the understanding of the molecular details behind this key regulatory network. Glycans attached to the death receptors such as CD95 and TRAIL-Rs, either alone or in a complex with galectins, might promote or inhibit apoptotic signals. However, we have just started to decode the functions of galectins in the modulation of extrinsic and intrinsic apoptosis. In this work, we have discussed the current understanding of the glycosylation-galectin regulatory network in CD95- as well as TRAIL-R-induced apoptosis and therapeutic strategies based on targeting galectins in cancer.

Selenium Protects Retinal Cells from Cisplatin-Induced Alterations in Carbohydrate Residues.

BACKGROUND: Investigate alterations in the expression and localization of carbohydrate units in rat retinal cells exposed to cisplatin toxicity. AIMS: The aim of the study was to evaluate putative protective effects of selenium on retinal cells subjected to cisplatin. STUDY DESIGN: Animal experiment. METHODS: Eighteen healthy Wistar rats were divided into three equal groups: 1. Control, 2. Cisplatin and 3. Cisplatin+selenium groups. After anesthesia, the right eye of each rat was enucleated. RESULTS: Histochemically, retinal cells of control groups reacted with α-2,3-bound sialic acid-specific Maackia amurensis lectin (MAA) strongly, while cisplatin reduced the staining intensity for MAA. However, selenium administration alleviated the reducing effect of cisplatin on the binding sites for MAA in retinal cells. The staining intensity for N-acetylgalactosamine (GalNAc residues) specific Griffonia simplicifolia-1 (GSL-1) was relatively slight in control animals and cisplatin reduced this slight staining for GSL-1 further. Selenium administration mitigated the reducing effect of cisplatin on the binding sites for GSL-1. A diffuse staining for N-acetylglucosamine (GlcNAc) specific wheat germ agglutinin (WGA) was observed throughout the retina of the control animals. In particular, cells localized in the inner plexiform and photoreceptor layers are reacted strongly with WGA. Compared to the control animals, binding sites for WGA in the retina of rats given cisplatin were remarkably decreased. However, the retinal cells of rats given selenium reacted strongly with WGA. CONCLUSION: Cisplatin reduces α-2,3-bound sialic acid, GlcNAc and GalNAc residues in certain retinal cells. However, selenium alleviates the reducing effect of cisplatin on carbohydrate residues in retinal cells.

Lycopene Ameliorates Experimental Colitis in Rats via Reducing Apoptosis and Oxidative Stress.

Inflammatory bowel disease (IBD) is an inflammatory disorder involving colitis. Lycopene is a naturally occurring carotenoid that has attracted considerable attention as a potential chemopreventive agent. The impact of lycopene on colitis is currently unknown. The aim of this study was to investigate the protective effects of lycopene in a rat model of colitis induced by acetic acid. The animals were randomly divided into the following five groups: the control group, colitis group, colitis + sulfasalazine group as a positive control group, colitis + lycopene and lycopene groups. Colonic mucosal injury was assessed by biochemical and histopathological examinations. Malondialdehyde (MDA), superoxide dismutase (SOD) activity, total antioxidant status (TAS), ceruloplasmin (CPN), total sialic acid and iron (Fe) levels were evaluated in blood samples. MDA, SOD, TAS and DNA fragmentation levels were also measured in colon tissues. MDA (p < 0.05), total sialic acid (p < 0.05) and DNA fragmentation levels (p < 0.01) were significantly higher, and the activity of the antioxidant enzyme were lower in the colitis group than in the control group. Treatments with lycopene in the colitis decreased MDA, total sialic acid and DNA fragmentation levels, while SOD activity (p < 0.05), TAS (in colon p < 0.05; in serum p < 0.01), CPN (p < 0.05) and Fe levels (p < 0.05) were significantly increased. The histopathological evaluation also confirmed the foregoing findings. Treatment with lycopene ameliorated the biochemical and pathological alterations caused by colitis. The results obtained in this study indicate that lycopene may exert protective effects in experimental colitis and might, therefore, be useful for treatment of IBD.

The protective effect of curcumin administration on carbon tetrachloride (CCl4)-induced nephrotoxicity in rats.

BACKGROUND: The aim of the present study was to examine the protective effect of curcumin (CUR) on carbon tetrachloride (CCl4)-induced nephrotoxicity to evaluate the detailed mechanisms by which CUR exerts its protective action. METHODS: Thirty male Wistar-Albino rats weighing 250-300 g were randomly divided into three groups: administrations of olive oil (control, po), CCl4 (0.5mg/kg in olive oil sc) every other day for 3 weeks, and CCl4 (0.5mg/kg in olive oil sc) plus CUR (200mg/kg) every day for 3 weeks. RESULTS: Administration of CCl4 significantly (p<0.001) increased the levels of renal function test such as creatinine and blood urea nitrogen (BUN). Furthermore, treatment of CCl4 significantly elevated the oxidant status of renal tissues while decreasing its anti-oxidant status (p<0.001). CUR displayed a renal protective effect as evident by significant decrease in inflammation and apoptosis during histopathological examination. The administration of CCl4 resulted in an increase in malondialdehyde (MDA) production due to an increase in membrane lipid peroxidation; however, the administration of CUR attenuated this, probably via its antioxidant and free radical scavenging properties. CONCLUSION: The finding of our study indicates that CUR may have an important role to play in protecting the kidney from oxidative insult.

Comparison of pre-treatment and post-treatment use of selenium in retinal ischemia reperfusion injury.

AIM: To investigate the effects of selenium in rat retinal ischemia reperfusion (IR) model and compare pre-treatment and post-treatment use. METHODS: Selenium pre-treatment group (n=8) was treated with intraperitoneal (i.p.) selenium 0.5 mg/kg for 7d and terminated 24h after the IR injury. Selenium post-treatment group (n=8) was treated with i.p. selenium 0.5 mg/kg for 7d after the IR injury with termination at the end of the 7d period. Sham group (n=8) received i.p. saline injections identical to the selenium volume for 7d with termination 24h after the IR injury. Control group (n=8) received no intervention. Main outcome measures were retina superoxide dismutase (SOD), glutathione (GSH), total antioxidant status (TAS), malondialdehyde (MDA), DNA fragmentation levels, and immunohistological apoptosis evaluation. RESULTS: Compared to the Sham group, selenium pre-treatment had a statistical difference in all parameters except SOD. Post-treatment selenium also resulted in statistical differences in all parameters except the MDA levels. When comparing selenium groups, the pre-treatment selenium group had a statistically higher success in reduction of markers of cell damage such as MDA and DNA fragmentation. In contrast, the post-selenium treatment group had resulted in statistically higher levels of GSH. Histologically both selenium groups succeeded to limit retinal thickening and apoptosis. Pre-treatment use was statistically more successful in decreasing apoptosis in ganglion cell layer compared to post-treatment use. CONCLUSION: Selenium was successful in retinal protection in IR injuries. Pre-treatment efficacy was superior in terms of prevention of tissue damage and apoptosis.

Inhibitory effect of sub-conjunctival tocilizumab on alkali burn induced corneal neovascularization in rats.

BACKGROUND: To evaluate the effects of sub-conjunctivally applied interleukin-6 receptor (IL-6R) antibody (tocilizumab) on alkali burn induced corneal neovascularization (CNV) in rats. METHODS: Alkali burn induced corneal neovascularization was created in 24 right eyes of 24 rats. The rats were then randomized into 2 groups. Group 1 received sub-conjunctival injection of 4 mg/0.2 ml tocilizumab and Group 2 received sub-conjunctival injection of 0.2 ml normal saline at the 5th day of alkali burn. The corneal surface area invaded with neovascular vessels were calculated on photographs. The rats were sacrificed and the corneas were excised at the15th day. The corneal specimens were stained with hemotoxylin-eosin to evaluate tissue morphology and with Willebrand factor (vWF) to evaluate microvascular structures immunohistochemically. Vascular endothelial growth factor (VEGF) expression was analyzed by ELISA. RESULTS: The percent area of CNV was 26.9% in Group 1 and 56.5% in Group 2 (p < 0.001). The histological evaluation showed that the corneal structures were not visibly altered by sub-conjuntival tocilizumab injection. Group 1 showed significantly lower corneal inflammation score than Group 2 (p < 0.001). The number of vessels stained with vWF were significantly higher in Group 2 than Group 1 (15.23 and 5.46, respectively; p < 0.001). ELISA analyses showed that corneal VEGF levels were significantly lower in Group 1 compared to Group 2 (p = 0.013) CONCLUSION: The present data demonstrated first time the beneficial effects of sub-conjunctival tocilizumab on decreasing CNV in alkali burn model of the rat cornea. Further studies are warranted to confirm these findings for the clinical application.

Protective effect of 2-APB on testicular ischemia-reperfusion injury in rats.

PURPOSE: We performed biochemical and histopathological evaluations to assess the effects of 2-APB on ischemia-reperfusion induced testicular damage. MATERIALS AND METHODS: A total of 28 rats were randomly divided into 4 groups, including sham treated, ischemia-reperfusion, ischemia-reperfusion plus 2 mg/kg 2-APB and ischemia-reperfusion plus 4 mg/kg 2-APB. Testicular tissue superoxide dismutase, glutathione, malondialdehyde, total antioxidant capacity and DNA fragmentation levels were determined. Testicular tissue samples were examined by histopathology and TUNEL staining. RESULTS: Mean superoxide dismutase, total antioxidant capacity and glutathione were significantly higher in the sham treated group than in the ischemia-perfusion group (p <0.05). Mean malondialdehyde and DNA fragmentation levels were significantly lower in the sham treated group than in the ischemia-reperfusion group (p <0.05). After 2-APB treatment superoxide dismutase, total antioxidant capacity and glutathione were significantly increased but malondialdehyde and DNA fragmentation levels were significantly decreased compared to the ischemia-reperfusion group (p <0.05). The number of TUNEL positive cells was significantly lower in the 2-APB treatment groups than in the ischemia-reperfusion group (p <0.05). CONCLUSIONS: In rats 2-APB reduced the oxidative stress and apoptosis caused by testicular ischemia-reperfusion injury. The testicular protective effect of 2-APB appears to be mediated through its antiapoptotic and antioxidative effects.

Protective effect of 2-aminoethyl diphenylborinate on acute ischemia-reperfusion injury in the rat kidney.

BACKGROUND: To investigate the protective effect of 2-aminoethyl diphenylborinate (2-APB) against ischemia-reperfusion (I/R) injury in the rat kidney by an experimental study. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were randomly divided into the following three groups: (1) sham group, (2) I/R group, and (3) I/R + 2-APB group. Renal I/R injury was induced by clamping the left renal pedicle for 45 min after right nephrectomy, followed by 3 h of reperfusion. The therapeutic agent 2-APB was administered intravenously at a dose of 2 mg/kg 10 min before renal ischemia. Glutathione, superoxide dismutase, total antioxidant capacity, malondialdehyde, tumor necrosis factor α, interleukin 6, aspartate aminotransferase, alanine aminotransferase, and creatinine levels were measured from blood samples, and the rats were sacrificed subsequently. Tissue samples were scored histopathologically. Visualization of apoptotic cells was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling staining method. RESULTS: 2-APB significantly reduced serum malondialdehyde, tumor necrosis factor α, interleukin 6, aspartate aminotransferase, alanine aminotransferase, and creatinine levels in the I/R injury group. However, glutathione, superoxide dismutase, and total antioxidant capacity levels increased significantly. Histopathologic scores were significantly better and the rate of apoptosis was lower in the 2-APB group. CONCLUSIONS: 2-APB reduces oxidative stress and damage caused by renal I/R injury. The results of this study demonstrate that 2-APB can be used as an effective agent against I/R injury in the kidney.

Effects of xylene and formaldehyde inhalations on oxidative stress in adult and developing rats livers.

In this study, it was aimed to demonstrate the possible oxidative stress caused by exposure of xylene and formaldehyde (HCHO) on liver tissue, and on body and liver weights in adult as well as developing rats. The rats (96 female Sprague-Dawley) were randomly divided into four groups: embryonic day 1 (Group 1), 1-day-old infantile rats (Group 2), 4-week-old rats (Group 3) and adult rats (Group 4). The animals were exposed to gases of technical xylene (300 ppm), HCHO (6 ppm) or technical xylene + HCHO (150 ppm + 3 ppm), 8 hours per day for 6 weeks. Superoxide dismutase (SOD) and catalase (CAT) activities, and glutathione (GSH) and malondialdehyde (MDA) levels were evaluated. In addition, body and liver weights were determinated. Compared to the control animals, body and liver weights were decreased in the embryonic day 1 group (P < 0.001, P < 0.01, respectively) and the 1-day-old infantile group (P < 0.001). Liver weight was increased in the 4-week-old group (P < 0.01). SOD activities were decreased in the 4-week-old rats exposed to HCHO (P < 0.01). CAT activities increased in the embryonic day 1 group (P < 0.05). GSH levels were decreased in the 1-day-old infantile group (P < 0.01), and MDA levels was increased in the embryonic day 1 group (P < 0.05) as compared with the respective control groups. As to GSH and MDA levels in adult and 4-week-old animals, no statistically significant differences were observed (P > 0.05). The present study indicates that exposures to xylene, HCHO and a mixture of them are toxic to liver tissue, and developing female rats are especially more adversely affected. Furthermore, the results of this study show that adult female rats could better tolerate the adverse effects of these toxic gases.