The bipyridyl herbicide paraquat induces proteasome dysfunction in human neuroblastoma SH-SY5Y cells

Paraquat (PQ) is suspected to be an environmental risk factor for Parkinson's disease (PD). A strong correlation between exposure to paraquat and the occurrence of PD was reported in Canada, Taiwan, and the United States. This correlation is supported by in vivo work showing that paraquat produces dopaminergic pathogenesis. In particular, paraquat forms abnormal protein aggregates in dopaminergic neurons of mice. However, it is not clear how paraquat produces this pathology. Given that proteasome dysfunction induces aberrant protein aggregation, it was hypothesized that paraquat induces proteasome dysfunction. To explore this possibility, proteasome activity and some factors possibly contributing to proteasome dysfunction were investigated in dopaminergic SY5Y cells treated with paraquat. Furthermore, levels of alpha-synuclein and ubiquitin-conjugated proteins were measured to test whether paraquat induces protein accumulation in SY5Y cells. Results showed that at a concentration of paraquat that reduced viability by about 60% at 48 h (0.5 mM) loss of proteasome activity occurred. In addition, the cells showed decreased ATP levels and reduced mitochondrial complex V activity. These changes were significant 24 h after treatment with paraquat. Furthermore, paraquat-treated cells showed decreased protein levels of proteasome 19S subunits, but not 20S alpha or beta subunits, suggesting that the effects observed were not the result of general cytotoxicity. Paraquat also increased levels of alpha-synuclein and ubiquitinated proteins, suggesting that paraquat-induced proteasome dysfunction leads to aberrant protein accumulation. Taken together, these findings support the hypothesis that paraquat impairs proteasome function in SY5Y cells.     

The bipyridyl herbicide paraquat produces oxidative stress-mediated toxicity in human neuroblastoma SH-SY5Y cells: relevance to the dopaminergic pathogenesis

Paraquat (PQ) is a cationic nonselective bipyridyl herbicide widely used to control weeds and grasses in agriculture. Epidemiologic studies indicate that exposure to pesticides can be a risk factor in the incidence of Parkinson's disease (PD). A strong correlation has been reported between exposure to paraquat and PD incidence in Canada, Taiwan, and the United States. This correlation is supported by animal studies showing that paraquat produces toxicity in dopaminergic neurons of the rat and mouse brain. However, it is unclear how paraquat triggers toxicity in dopaminergic neurons. Based on the prooxidant properties of paraquat, it was hypothesized that paraquat may induce oxidative stress-mediated toxicity in dopaminergic neurons. To explore this possibility, dopaminergic SH-SY5Y cells were treated with paraquat, and several biomarkers of oxidativestress were measured. First, a specific dopamine transporter inhibitor GBR12909 significantly protected SY5Y cells against the toxicity of paraquat, indicating that paraquat exerts its toxicity by a mechanism involving the dopamine transporter (DAT). Second, paraquat increased intracellular levels of reactive oxygen species (ROS), but decreased the levels of glutathione. Third, paraquat inhibited glutathione peroxidase activity, but did not affect glutathione reductase activity. On the other hand, paraquat increased GST activity by 24 h, after which GST activity returned to the control value at 48 h. Fourth, paraquat dissipated mitochondrial transmembrane potential (MTP). Fifth, paraquat produced increases of malondialdehyde (MDA) and protein carbonyls, as well as DNA fragmentation, indicating oxidative damage to major cellular components. Sixth, paraquat increased the protein level of heme oxygenase-1 (HO-1). Taken together, these findings verify our hypothesis that paraquat produces oxidative stress-mediated toxicity in SH-SY5Y cells. Thus, current findings suggest that paraquat may induce the pathogenesis of dopaminergic neurons through oxidative stress.     

Paraquat-induced apoptosis in human neuroblastoma SH-SY5Y cells: involvement of p53 and mitochondria

The herbicide paraquat is a suspected etiologic factor in the development of Parkinson's disease (PD). Paraquat was therefore used to reproduce Parkinsonian syndromes in lab animals, in which it produces dopaminergic pathogenesis. However, the factors or mechanisms by which paraquat kills dopaminergic neurons are not fully understood. Based on reported evidence that paraquat increases p53 protein levels and inhibits mitochondrial function, it was hypothesized that paraquat induces cell death in dopaminergic neurons through a mechanism in which p53 and mitochondrial apoptotic pathway are linked. To explore this possibility, dopaminergic SY5Y cells were treated with paraquat for 48 h and p53 responses were investigated, as well as biomarkers of the mitochondrial intrinsic pathway of apoptosis. Paraquat significantly increased protein levels of p53 and one of its target genes, Bax. By 24 h, paraquat decreased mitochondrial complex I activity and mitochondrial transmembrane potential and induced the release of cytochrome c from mitochondria. In addition, paraquat increased the activities of caspases 9 and 3. Finally, nuclear condensation and DNA fragmentation occurred 48 h after treatment. The decrease of mitochondrial functions, the release of cytochrome c, the increase of caspase 9 and 3 activities, and DNA damage that were produced by paraquat were inhibited by a specific p53 inhibitor, pifithrin-alpha. These findings support the conclusion that paraquat produced apoptosis in SY5Y cells through the mitochondrial intrinsic pathway associated with p53.     

Regulation of dopaminergic neuronal death by endogenous dopamine and proteasome activity

Parkinson disease is one of the most common neurodegenerative disorders and is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Although endogenous dopamine itself could serve as a vulnerability factor for dopaminergic neurons, the mechanism by which dopamine contributes to dopaminergic neuronal death remains unknown. In addition, although a decrease in proteasome activity was found in patients with sporadic Parkinson disease, the relationship between the ubiquitin-proteasome system and dopaminergic neuronal death remains to be elucidated. Here we provide an overview of the roles of endogenous dopamine and proteasome activity in dopaminergic cell death. Treatment of catecholaminergic PC12 cells with the herbicide paraquat, a potential risk factor for the development of Parkinson disease, induced an increase in dopamine content, and depletion of intracellular dopamine suppressed paraquat-induced cytotoxicity. Although glutathione, which scavenged dopamine oxidation intermediate, provided almost complete protection against dopamine-mediated toxicity, catalase provided only partial protection against cell death caused by dopamine. These data suggest that the generation of dopamine oxidation intermediate, rather than that of reactive oxygen species, plays a pivotal role in dopamine-induced toxicity. Moreover, treatment with paraquat induced a decrease in proteasome activity, and proteasome inhibition suppressed dopamine-mediated cytotoxicity. Suppression of proteasome activity stimulated the NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, and elevated γ-glutamylcysteine synthetase mRNA and glutathione content. Furthermore, suppression of the paraquat-induced increase in gluthathione content exacerbated paraquat toxicity. These results suggest that the reduction of proteasome activity may be involved in cellular defense mechanisms against dopamine-mediated paraquat toxicity.     

DJ-1 mediates paraquat-induced dopaminergic neuronal cell death

There are two causes of Parkinson's disease (PD): environmental insults and genetic mutations of PD-associated genes. Environmental insults and genetic mutations lead to mitochondrial dysfunction, and a combination of mitochondrial dysfunction and increased oxidative stress in dopaminergic neurons is thought to contribute to the pathogenesis of PD. Among the PD-associated genes, DJ-1 acts as a redox sensor for oxidative stress and has been also proposed to maintain mitochondrial complex I activity.To understand molecular functions of DJ-1 in the cell, we have generated DJ-1 null cells from the DJ-1(−/−) mouse embryos. Using these null cells, we investigated the susceptibility to an environmental toxin, paraquat, which is known to inhibit mitochondrial complex I. Interestingly, we found that DJ-1 null cells showed a resistance to paraquat-induced apoptosis, including reduced poly (ADP-ribose) polymerase and procaspase-3. Also DJ-1 null cells generated less superoxide than SN4741 cells by paraquat treatment. Consistent with the reduced paraquat sensitivity, DJ-1 null cells showed reduced complex I activity, which was partially rescued by ectopic DJ-I expression. In summary, our results suggest that DJ-1 is critical to maintain mitochondrial complex I and complex I could be a key target in interaction of paraquat toxicity and DJ-1 for giving rise to PD.     

Emodin protected against synaptic impairment and oxidative stress induced by fluoride in SH‐SY5Y cells by modulating ERK1/2/Nrf2/HO‐1 pathway

Excessive fluoride exposure contributes to neurotoxic effects. Emodin exhibits antioxidative functions in the central nervous system (CNS); however, its neuroprotective mechanism against fluoride remains to be elucidated. Our aim was to explore the neuroprotective efficacy and the possible mechanisms of emodin. In our study, synaptic proteins and oxidative stress damage were examined after human neuroblastoma SH‐SY5Y cells were treated with high doses of NaF for 24 hours. Moreover, pretreatment with emodin was used to shed light on the neuroprotective effects in NaF‐induced toxicity in SH‐SY5Y cells. We found that NaF significantly lowered the protein expressions of SNAP 25, synaptophysin and PSD 95 in SH‐SY5Y cells. In addition, NaF exposure increased the protein expression of p‐ERK1/2 and decreased the protein expressions of Nrf2 and HO‐1, as well as facilitated increasing ROS, 4‐hydroxynonenal (4‐HNE), and 8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG). Pretreatment with emodin significantly recovered these alterations caused by NaF. These data implied that the neuroprotective effects of emodin and pointed to the promising utilization for protecting against neurotoxicity induced by fluoride.     

Glutathione biosynthesis via activation of the nuclear factor E2-related factor 2 (Nrf2)--antioxidant-response element (ARE) pathway is essential for neuroprotective effects of sulforaphane and 6-(methylsulfinyl) hexyl isothiocyanate

Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress-induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H(2)O(2)- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2-related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-L-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H(2)O(2) and paraquat. Moreover, exposure to L-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.     

Activation of c-Jun N-terminal protein kinase is a common mechanism underlying paraquat- and rotenone-induced dopaminergic cell apoptosis.

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the substantia nigra of the brain. Although the underlying causes are not well characterized, epidemiological studies suggest an elevated risk of PD with occupational pesticide exposure. Here, we utilized pheochromocytoma (PC) 12 and SH-SY5Y cells as well as rat primary cultured dopaminergic neurons to investigate mechanisms for dopaminergic cell death induced by paraquat and rotenone, pesticides that are used to model PD in rodents. Both paraquat and rotenone induce selective loss of dopaminergic neurons in primary cultures. We discovered that paraquat induces apoptosis in PC12 cells but not in SH-SY5Y cells, while rotenone exposure causes apoptosis in SH-SY5Y cells but not in PC12 cells. The selective ability of paraquat and rotenone to induce apoptosis in different cell lines correlates with their ability to activate c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinases. Furthermore, JNK and p38 are required for rotenone-induced apoptosis in SH-SY5Y cells (K. Newhouse et al., 2004, Toxicol. Sci. 79, 137-146) as well as primary neurons, and for paraquat-induced apoptosis in PC12 cells. However, JNK but not p38 plays a role in paraquat-induced loss of primary cultured dopaminergic neurons. Our data identify JNK activation as a common mechanism underlying dopaminergic cell death induced by both paraquat and rotenone in model cell lines and primary cultures.     

NF-κB靶向性哑铃形“圈套”寡核苷酸对海马神经元及神经元样细胞中COX-2表达的影响

目的设计合成靶向性NF-κB的哑铃形圈套ODNs,并检测其对神经元样细胞及海马神经元中NF-κB及环氧化酶-2(COX-2)的抑制作用。方法采用NF-κB靶向性哑铃形圈套ODNs转染培养的PC12细胞,用Western blot方法对PC12细胞内NF-κB和COX-2的蛋白表达进行分析;转染培养的海马神经元再经LPS刺激后,用RT-PCR法检测COX-2mRNA。结果经靶向性NF-κB哑铃形圈套ODNs处理的PC12细胞中NF-κB的表达明显降低(P<0.01),同时伴有COX-2表达明显减少(P<0.01);海马神经元中COX-2mR-NA的表达明显降低(P<0.01)。结论靶向性NF-κB的哑铃形圈套ODNs可抑制神经元样细胞及神经细胞中NF-κB和COX-2的表达,且NF-κB对COX-2具有调控作用。     

Chloroquine attenuates paraquat-induced lung injury in mice by altering inflammation,oxidative stress and fibrosis

Paraquat is one of the most extensively used herbicides and has high toxicity for humans and animals. However, there is no effective treatment for paraquat poisoning. The aim of the present study was to evaluate the effects of chloroquine on paraquat-induced lung injury in mice. Mice received a single intraperitoneal injection of paraquat and a daily intraperitoneal injection of the indicated dosages of chloroquine or dexamethasone. The histological changes, inflammation and oxidative stress in the lungs were examined at day 3, and the degree of pulmonary fibrosis was examined at day 28. H&E staining showed that chloroquine markedly attenuated lung injury induced by paraquat. In addition, the inflammatory responses induced by paraquat were inhibited after treatment with chloroquine, as indicated by the decreased number of leukocytes, the reduced levels of TNF-α, IL-1β and IL-6 in the bronchoalveolar lavage fluid, the reduced NO content, and downregulation of iNOS expression in lung tissues. No different effect was found between high-dose chloroquine and dexamethasone. Additionally, the treatment with chloroquine increased the activity of SOD and decreased the level of MDA in the lung tissues. The expressions of the anti-oxidative proteins, Nrf2, HO-1 and NQO1, were also upregulated by chloroquine treatment. The high-dose chloroquine was more effective than dexamethasone in its anti-oxidation ability. Finally, the results of Masson's staining illustrated that chloroquine markedly attenuated fibrosis in the paraquat-exposed lungs. Immunohistochemistry staining showed that the expressions of the pro-fibrotic proteins TGF-β and α-SMA were downregulated after treatment with chloroquine. In conclusion, chloroquine effectively attenuated paraquat-induced lung injury in mice.     

Effect of genetic variation on human cytochrome p450 reductase-mediated paraquat cytotoxicity.

Paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride) is a widely used herbicide and is highly toxic to human and animals. The mechanisms of paraquat toxicity involve the generation of superoxide anion through the process of redox cycling. NADPH-cytochrome P450 oxidoreductase (POR) has been reported to be a major enzyme for one-electron reduction of paraquat that initiates the redox cycling. Recently, a total of six missense variants of human POR have been identified in patients with discorded steroidogenesis. However, the effect of these genetic variations on POR-mediated paraquat toxicity is not known. Using the Flp-In Chinese hamster ovary (CHO) cells stably expressing either mouse or human POR and the cells with POR knockdown by siRNA, we confirmed that POR is responsible for paraquat-induced cytotoxicity. We further used this validated system to compare paraquat-induced toxicity among the cells that stably expressed wild-type human POR and its natural variants. While there was no difference in paraquat-induced toxicity between the cells expressing wild-type human POR and the Cys569Tyr variant, the toxicity in cells expressing all the other variants (Tyr181Asp, Ala287Pro, Arg457His, Val492Glu, and Val608Phe) was significantly decreased. Our results provide further evidence on the important role of POR in paraquat-induced toxicity and suggest that individuals carrying the functional variant POR alleles may have an altered susceptibility to paraquat exposure.     

Cryptochlorogenic acid attenuates LPS-induced inflammatory response and oxidative stress via upregulation of the Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages

Phenolic acids are found in natural plants, such as caffeic acid, rosmarinic acid, and chlorogenic acid. They have long been used as pharmacological actives, owing to their anti-inflammatory and antioxidant activities. Cryptochlorogenic acid (CCGA) is a special isomer of chlorogenic acid; the pharmacological effects and related molecular mechanisms of CCGA have been poorly reported. In the present study, the antioxidant and anti-inflammatory effects of CCGA in RAW 264.7 macrophages and the underlying mechanisms were investigated. The results revealed that CCGA dose-dependently inhibited LPS-induced production of NO, TNF-α, and IL-6 and blocked iNOS, COX-2, TNF-α, and IL-6 expressions. CCGA also significantly increased the GSH/GSSG ratio and SOD activity and reduced the MDA level. Moreover, CCGA suppressed the nuclear translocation of NF-κB by hindering the phosphorylation of IκB kinase (IKK) and degrading IκB. It also downregulated the phosphorylation of MAPKs. Our results indicated that CCGA significantly inhibited NF-κB activation by controlling the expression of pro-inflammatory factors and promoting the nuclear transfer of Nrf2. In conclusion, CCGA could attenuate LPS-induced inflammatory symptoms by modulating NF-κB/MAPK signaling cascades and inhibit LPS-induced oxidative stress via Nrf2 nuclear translocation.     

原花青素对白介素-1β诱导的A549细胞环氧合酶-2 mRNA转录的抑制作用

目的研究原花青素对白介素-1β(IL-1β)诱导的人肺癌细胞A549中环氧合酶-2(COX-2)mRNA转录的抑制机制。方法采用RT-PCR法测定原花青素对IL-1β诱导的A549细胞中COX-2 mRNA转录的影响,采用Western blot和免疫组化法考察原花青素对IL-1β诱导的A549细胞核转录因子κB(NF-κB)亚基p65(NF-κB/p65)及NF-κB抑制性蛋白(I-κB)表达的抑制作用。结果原花青素对A549细胞中COX-2 mRNA的转录有较强抑制作用,抑制NF-κB/p65的表达及I-κB的降解。结论原花青素可能是通过抑制NF-κB/p65的表达及I-κB的降解而抑制COX-2mRNA的转录。     

Sulforaphane protects against cytokine- and streptozotocin-induced β-cell damage by suppressing the NF-κB pathway

Sulforaphane (SFN) is an indirect antioxidant that protects animal tissues from chemical or biological insults by stimulating the expression of several NF-E2-related factor-2 (Nrf2)-regulated phase 2 enzymes. Treatment of RINm5F insulinoma cells with SFN increases Nrf2 nuclear translocation and expression of phase 2 enzymes. In this study, we investigated whether the activation of Nrf2 by SFN treatment or ectopic overexpression of Nrf2 inhibited cytokine-induced β-cell damage. Treatment of RIN cells with IL-1β and IFN-γ induced β-cell damage through a NF-κB-dependent signaling pathway. Activation of Nrf2 by treatment with SFN and induction of Nrf2 overexpression by transfection with Nrf2 prevented cytokine toxicity. The mechanism by which Nrf2 activation inhibited NF-κB-dependent cell death signals appeared to involve the reduction of oxidative stress, as demonstrated by the inhibition of cytokine-induced H₂O₂ production. The protective effect of SFN was further demonstrated by the restoration of normal insulin secreting responses to glucose in cytokine-treated rat pancreatic islets. Furthermore, pretreatment with SFN blocked the development of type 1 diabetes in streptozotocin-treated mice.     

Medical management of paraquat ingestion

Poisoning by paraquat herbicide is a major medical problem in parts of Asia while sporadic cases occur elsewhere. The very high case fatality of paraquat is due to inherent toxicity and lack of effective treatments. We conducted a systematic search for human studies that report toxicokinetics, mechanisms, clinical features, prognosis and treatment. Paraquat is rapidly but incompletely absorbed and then largely eliminated unchanged in urine within 12-24 h. Clinical features are largely due to intracellular effects. Paraquat generates reactive oxygen species which cause cellular damage via lipid peroxidation, activation of NF-κB, mitochondrial damage and apoptosis in many organs. Kinetics of distribution into these target tissues can be described by a two-compartment model. Paraquat is actively taken up against a concentration gradient into lung tissue leading to pneumonitis and lung fibrosis. Paraquat also causes renal and liver injury. Plasma paraquat concentrations, urine and plasma dithionite tests and clinical features provide a good guide to prognosis. Activated charcoal and Fuller's earth are routinely given to minimize further absorption. Gastric lavage should not be performed. Elimination methods such as haemodialysis and haemoperfusion are unlikely to change the clinical course. Immunosuppression with dexamethasone, cyclophosphamide and methylprednisolone is widely practised, but evidence for efficacy is very weak. Antioxidants such as acetylcysteine and salicylate might be beneficial through free radical scavenging, anti-inflammatory and NF-κB inhibitory actions. However, there are no published human trials. The case fatality is very high in all centres despite large variations in treatment.     

Melatonin reduces paraquat-induced genotoxicity in mice

The protection afforded by melatonin against paraquat-induced genotoxicity in both bone marrow and peripheral blood cells of mice was tested using micronuclei as an index of induced chromosomal damage. Melatonin (2 mg/kg) or an equal volume of saline was injected i.p. into mice 30 min prior to the i.p. administration of paraquat (two injections of 15 mg/kg; the paraquat injections were given with a 24 h interval) and thereafter at 6 h intervals to the conclusion of the study (72 h). Using fluorescence microscopy, the number of micronuclei in polychromatic erythrocytes (MN-PCE) per 2000 PCE (1000 PCE/slide) per mouse was counted both in blood and bone marrow, and the ratio of PCE to normochromatic erythrocytes (NCE) (PCE/NCE) was calculated. Paraquat treatment increased the number of MN-PCE at 24, 48, and 72 h, both in peripheral blood and bone marrow cells, while no differences were observed in the PCE/NCE ratio. Melatonin inhibited the paraquat-induced increase in MN-PCE by more than 50% at 48 and 72h. Paraquat toxicity is believed to be due to free radical generation. Since melatonin is known to be an efficient free radical scavenger, it is concluded that melatonin's protection against paraquat-induced genotoxicity is mediated, at least in part, by its free radical scavenging activity.     

Astrocyte mediated protection of fetal cerebral cortical neurons from rotenone and paraquat

Primary cultures of fetal rat cortical neurons and astrocytes were used to test the hypothesis that astrocyte-mediated control of neuronal glutathione (GSH) is a potent factor in neuroprotection against rotenone and paraquat. In neurons, rotenone (0.025-1 μM) for 4 and 24 h decreased viability as did paraquat (2-100 μM). Rotenone (30 nM) decreased neuronal viability and GSH by 24% and 30%, while ROS were increased by 56%. Paraquat (30 μM) decreased neuronal viability and GSH by 36% and 70%, while ROS were increased by 23%. When neurons were co-cultured with astrocytes, their GSH increased 1.5 fold and 5 fold at 12 and 24 h. Co-culturing with astrocytes blocked neuronal death and damage by rotenone and paraquat. Astrocyte-mediated neuroprotection was dependent on the activity of components of the γ-glutamyl cycle. These studies illustrate the importance of astrocyte-mediated glutathione homeostasis for protection of neurons from rotenone and paraquat and the role of the γ-glutamyl cycle in this neuroprotection.     

Betanin attenuates paraquat-induced liver toxicity through a mitochondrial pathway

We attempted to determine whether betanin (from natural pigments) that has anti-oxidant properties would be protective against paraquat-induced liver injury in Sprague–Dawley rats. Paraquat was injected intraperitoneally into rats to induce liver toxicity. The rats were randomly divided into four groups: a control group, a paraquat group, and two groups that received betanin at doses of 25 and 100 mg/kg/day three days before and two days after they were administered paraquat. We evaluated liver histopathology, serum liver enzymatic activities, oxidative stress, cytochrome P450 (CYP) 3A2 mRNA expression, and mitochondrial damage. The rats that were injected with paraquat incurred liver injury, evidenced by histological changes and elevated serum aspartate aminotransferase and alanine aminotransferase levels; paraquat also led to oxidative stress, an increase of cytochrome P450 3A2 mRNA expression, and mitochondrial damage, indicated by mitochondrial membrane swelling, reduced mitochondrial cytochrome C, and apoptosis-inducing factor protein levels. Pathological damage and all of the above mentioned markers were lesser in the animals treated with betanin than in those who received paraquat alone. Betanin had a protective effect against paraquat-induced liver damage in rats. The mechanism of the protection appears to be the inhibition of CYP 3A2 expression and protection of mitochondria.