Targeting HMGB1 inhibits T-2 toxin-induced neurotoxicity via regulation of oxidative stress, neuroinflammation and neuronal apoptosis.

T-2 toxin, a food-derived mycotoxin, has been identified as a neurotoxin. Nonetheless, T-2 toxin-induced neuroinflammation has never been revealed. As an important therapeutic target for inflammatory diseases and cancers, the role of high mobility group B1 (HMGB1) in mycotoxin-mediated neurotoxicity remains a mystery. In current study, we found that PC12 cells were sensitive to trace amounts of T-2 toxin less than 12 ng/mL, distinguished by decreased cell viability and increased release of lactate dehydrogenase (LDH). Oxidative stress and mitochondrial damage were observed in PC12 cells, manifested as accumulation of oxidative stress products, up-regulation of Nrf2/HO-1 pathway and decrease of mitochondrial membrane potential (MMP), leading to mitochondria-dependent apoptosis. Meanwhile, we first discovered that tiny amounts of T-2 toxin triggered neuroinflammation directly, including raising the expression and translocation of NF-κB and promoting secretion of proinflammatory cytokines such as TNF-α, IL-6, IL-8 and IL-1ß. Most interestingly, the increased of HMGB1 was detected both inside and outside the cells. Conversely, HMGB1 siRNA reduced T-2 toxin-mediated oxidative stress, apoptosis and neuroinflammatory outbreak, accompanied by lessened caspase-3 and caspase-9, and decreased secretion of pro-inflammatory cytokines. Taken together, T-2 toxin-stimulated PC12 cells simultaneously displayed apoptosis and inflammation, whereas HMGB1 played a critical role in these neurotoxic processes.

Curcumin Ameliorates Copper-Induced Neurotoxicity Through Inhibiting Oxidative Stress and Mitochondrial Apoptosis in SH-SY5Y Cells.

Impaired homeostasis of copper has been linked to different pathophysiological mechanisms in neurodegenerative diseases and oxidative injury has been proposed as the main mechanism. This study aims to use curcumin, a widely used antioxidative and anti-apoptotic agent, to exert the neuroprotective effect against copper in vitro and illuminate the underlying mechanism. The effect of curcumin was examined by using a cell counting kit-8 assay, flow cytometry, immunofluorescence, spectrophotometer, and western blot. Results revealed that after pretreatment with curcumin for 3 h, copper-induced toxicity and apoptosis show a significant decline. Further experiments showed that curcumin not only decreased the production of ROS and MDA but also increased the activities of the ROS scavenging enzymes SOD and CAT. Moreover, curcumin treatment alleviated the decrease in mitochondrial membrane potential and the nuclear translocation of cytochrome c induced by copper. The protein levels of pro-caspase 3, pro-caspase 9, and PARP1 were up-regulated and the Bax/Bcl-2 ratio was down-regulated in the presence of curcumin. Taken together, our study demonstrates that curcumin has neuroprotective properties against copper in SH-SY5Y cells and the potential mechanisms might be related to oxidative stress and mitochondrial apoptosis.

Olaquindox-Induced Liver Damage Involved the Crosstalk of Oxidative Stress and p53 In Vivo and In Vitro.

Olaquindox (OLA), a member of the quinoxaline-N,N-dioxide family, has been widely used as a growth-promoting feed additive and treatment for bacterial infections. The toxicity has been a major concern, and the precise molecular mechanism remains poorly understood. The present study was aimed at investigating the roles of oxidative stress and p53 in OLA-caused liver damage. In a mouse model, OLA administration could markedly cause liver injury as well as the induction of oxidative stress and activation of p53. Antioxidant N-acetylcysteine (NAC) inhibited OLA-induced oxidative stress and p53 activation in vivo. Furthermore, knockout of the p53 gene could significantly inhibit OLA-induced liver damage by inhibiting oxidative stress and the mitochondria apoptotic pathway, compared to the p53 wild-type liver tissue. The cell model in vitro further demonstrated that p53 knockout or knockdown in the HCT116 cell and L02 cell significantly inhibited cell apoptosis and increased cell viability, presented by suppressing ROS production, oxidative stress, and the Nrf2/HO-1 pathway. Moreover, loss of p53 decreased OLA-induced mitochondrial dysfunction and caspase activations, with the evidence of inhibited activation of phosphorylation- (p-) p38 and p-JNK and upregulated cell autophagy via activation of the LC3 and Beclin1 pathway in HCT116 and L02 cells. Taken together, our findings provided a support that p53 primarily played a proapoptotic role in OLA-induced liver damage against oxidative stress and mitochondrial dysfunction, which were largely dependent on suppression of the JNK/p38 pathway and upregulation of the autophagy pathway via activation of LC3 and Beclin1.

Identification of Plant Soot as Novel Safe Feed Additive: Evaluation of 90-Day Oral Toxicity and Prenatal Developmental Toxicity in Rats.

Plant soot, as a novel feed additive, could not only improve digestive function but also adsorb mycotoxins and inhibit bacterial infections. The subchronic toxicity and prenatal developmental effects of plant soot were studied for the first time. Our results indicated that there was no subchronic toxicity in the range of 2,000-50,000 mg/kg plant soot added in the feed, and there was no significant difference in reproductive function, embryo development, and teratogenicity between the pregnant rats exposed to 312.5, 1,250, and 5,000 mg/kg plant soot and the control group. The maximum no-observed effect level (NOEL) of supplemental dosage in feed could be set to 50,000 mg/kg, and the maximum intragastric NOEL could be set to 5,000 mg/kg, which preliminarily provided guidance on daily additive amount or clinical protocols for plant soot, as well as promoting the development and application of this harmless antibiotic substitutes.

Chloroquine ameliorates carbon tetrachloride-induced acute liver injury in mice via the concomitant inhibition of inflammation and induction of apoptosis.

This is the first study to investigate the hepatoprotective effect of CQ on acute liver injury caused by carbon tetrachloride (CCl4) in a murine model and the underlying molecular mechanisms. Ninety-six mice were randomly divided into the control (n = 8), CQ (n = 8), CCl4 (n = 40), and CCl4 + CQ (n = 40) treatment groups. In the CCl4 group, mice were intraperitoneally (i.p) injected with 0.3% CCl4 (10 mL/kg, dissolved in olive oil); in the CCl4 + CQ group, mice were i.p injected with CQ at 50 mg/kg at 2, 24, and 48 h before CCl4 administration. The mice in the control and CQ groups were administered with an equal vehicle or CQ (50 mg/kg). Mice were killed at 2, 6, 12, 24, 48 h post CCl4 treatment and their livers were harvested for analysis. The results showed that CQ pre-treatment markedly inhibited CCl4-induced acute liver injury, which was evidenced by decreased serum transaminase, aspartate transaminase and lower histological scores of liver injury. CQ pretreatment downregulated the CCl4-induced hepatic tissue expression of high-mobility group box 1 (HMGB1) and the levels of serum HMGB1 as well as IL-6 and TNF-α. Furthermore, CQ pre-treatment inhibited autophagy, downregulated NF-kB expression, upregulated p53 expression, increased the ratio of Bax/Bcl-2, and increased the activation of caspase-3 in hepatic tissue. This is the first study to demonstrate that CQ ameliorates CCl4-induced acute liver injury via the inhibition of HMGB1-mediated inflammatory responses and the stimulation of pro-apoptotic pathways to modulate the apoptotic and inflammatory responses associated with progress of liver damage.

Curcumin Attenuates Colistin-Induced Neurotoxicity in N2a Cells via Anti-inflammatory Activity, Suppression of Oxidative Stress, and Apoptosis.

Neurotoxicity is an unwanted side-effect seen in patients receiving therapy with the "last-line" polymyxin antibiotics. This is the first study to show that colistin-induced neurotoxicity in neuroblastoma-2a (N2a) cells gives rise to an inflammatory response involving the IL-1ß/p-IκB-α/NF-κB pathway. Pretreatment with curcumin at 5, 10, and 20 µM for 2 h prior to colistin (200 µM) exposure for 24 h, produced an anti-inflammatory effect by significantly down-regulating the expression of the pro-inflammatory mediators cyclooxygenase-2 (COX-2), phosphorylation of the inhibitor of nuclear factor-kappa B (NF-κB) (p-IκB)-α, and concomitantly NF-κB levels. Moreover, curcumin significantly decreased intracellular reactive oxygen species (ROS) production and increased the activities of the anti-ROS enzymes superoxide dismutase, catalase, and the intracellular levels of glutathione. Curcumin pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation, and subsequent apoptosis. Overall, our findings demonstrate for the first time, a potential role for curcumin for treating polymyxin-induced neurotoxicity through the modulation of NF-κB signaling and its potent anti-oxidative and anti-apoptotic effects.

ROS-mediated oligomerization of VDAC2 is associated with quinocetone-induced apoptotic cell death.

Quinocetone (QCT) has been approved and widely used as an animal feed additive in China since 2003. However, investigations indicate that QCT shows potential toxicity both in vitro and in vivo. Although voltage dependent anion channel 1 (VDAC1) involved in regulating QCT-induced apoptotic cell death has been established, the role of voltage dependent anion channel 2 (VDAC2) in QCT-induced toxicity remains unclear. In this study, we showed that QCT-induced cell death was coupled to VDAC2 oligomerization. Moreover, VDAC inhibitor 4, 4'-diisothiocyano stilbene-2, 2'-disulfonic acid (DIDS) alleviated QCT-induced cell death and VDAC2 oligomerization. Meanwhile, overexpression VDAC2 aggravated QCT-induced VDAC2 oligomerization. In addition, caspase inhibitor Z-VAD-FMK and reactive oxidative species (ROS) scavenger N-acetyl-l-cysteine (NAC) apparently blocked QCT-induced cell death and VDAC2 oligomerization. Finally, overexpression N-terminal truncated VDAC2 attenuated QCT-induced VDAC2 oligomerization but had no influence on its localization to mitochondria when comparing to the full length of VDAC2. Taken together, our results reveal that ROS-mediated VDAC2 oligomerization is associated with QCT-induced apoptotic cell death. The N-terminal region of VDAC2 is required for QCT-induced VDAC2 oligomerization.

Critical role of p21 on olaquindox-induced mitochondrial apoptosis and S-phase arrest involves activation of PI3K/AKT and inhibition of Nrf2/HO-1pathway.

Olaquindox, a quinoxaline 1,4-di-N-oxide, is known as an antibacterial agent and feed additive to treat bacterial infections and promote animal growth. However, the potential mechanism of toxicity is still unknown. The present study aims to explore the molecular mechanism of p21 on olaquindox-induced mitochondrial apoptosis and S-phase arrest in human hepatoma G2 cells (HepG2). As a result, olaquindox promoted production of ROS, suppressed the protein expression p21 in p53-independent way and phosphorylated p21. Meanwhile, olaquindox activated AKT and Nrf2/HO-1 pathway, up-regulated Bax/Bcl-2 ratio, disrupted mitochondrial membrane potential (MMP) and subsequently caused cytochrome c release and a cascade activation of caspase, eventually induced apoptosis. Olaquindox could induce S-phase arrest in HepG2 cells involved with the increase of Cyclin A, Cyclin E and CDK 2. Furthermore, knockdown of p21 decreased cell viability, enhanced oxidative stress, aggravated olaquindox-induced mitochondrial apoptosis and S-phase arrest involvement of activating PI3K/AKT and inhibiting Nrf2/HO-1 pathway. PI3K/AKT inhibitor (LY294002) and HO-1inhibitor (ZnPP-IX) both increased olaquindox-induced apoptosis and S-phase arrest. In conclusion, knockdown of p21 increased olaquindox-induced mitochondrial apoptosis and S-phase arrest through further activating PI3K/AKT and inhibiting Nrf2/HO-1pathway. Our study provided new insights into the molecular mechanism of olaquindox and shed light on the role of p21.

TCS2 Increases Olaquindox-Induced Apoptosis by Upregulation of ROS Production and Downregulation of Autophagy in HEK293 Cells.

Olaquindox, a feed additive, has drawn public attention due to its potential mutagenicity, genotoxicity, hepatoxicity and nephrotoxicity. The purpose of this study was to investigate the role of tuberous sclerosis complex (TSC2) pathways in olaquindox-induced autophagy in human embryonic kidney 293 (HEK293) cells. The results revealed that olaquindox treatment reduced the cell viability of HEK293 cells and downregulated the expression of TSC2 in a dose- and time-dependent manner. Meanwhile, olaquindox treatment markedly induced the production of reactive oxygen species (ROS), cascaded to autophagy, oxidative stress, and apoptotic cell death, which was effectively eliminated by the antioxidant N-acetylcysteine (NAC). Furthermore, overexpression of TSC2 attenuated olaquindox-induced autophagy in contrast to inducing the production of ROS, oxidative stress and apoptosis. Consistently, knockdown of TSC2 upregulated autophagy, and decreased olaquindox-induced cell apoptosis. In conclusion, our findings indicate that TSC2 partly participates in olaquindox-induced autophagy, oxidative stress and apoptosis, and demonstrate that TSC2 has a negative regulation role in olaquindox-induced autophagy in HEK293 cells.

Quinocetone induces mitochondrial apoptosis in HepG2 cells through ROS-dependent promotion of VDAC1 oligomerization and suppression of Wnt1/ß-catenin signaling pathway.

Quinocetone (QCT) has been used as an animal feed additive in China since 2003. However, investigations indicate that QCT has potential toxicity due to the fact that it shows cytotoxicity, genotoxicity, hepatotoxicity, nephrotoxicity and immunotoxicity in vitro and animal models. Although QCT-induced mitochondrial apoptosis has been established, the molecular mechanism remains unclear. This study was aimed to investigate the role of voltage-dependent anion channel 1 (VDAC1) oligomerization and Wnt/ß-catenin pathway in QCT-induced mitochondrial apoptosis. The results showed VDAC inhibitor 4, 4-diisothiocyano stilbene-2, 2-disulfonic acid (DIDS) partly compromised QCT-induced cell viability decrease (from 34.1% to 68.5%) and mitochondrial apoptosis accompanied by abating VDAC1 oligomerization, cytochrome c (Cyt c) release and the expression levels of cleaved caspase-9, -3 and poly (ADP-ribose) polymerase (PARP). Meanwhile, overexpression VDAC1 exacerbated QCT-induced VDAC1 oligomerization and Cyt c release. In addition, lithium chloride (LiCl), an activator of Wnt/ß-catenin pathway, markedly attenuated QCT-induced mitochondrial apoptosis by partly restoring the expression levels of Wnt1 and ß-catenin. Finally, reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) obviously blocked QCT-induced VDAC1 oligomerization and the inhibition of Wnt1/ß-catenin pathway. Taken together, our results reveal that QCT induces mitochondrial apoptosis by ROS-dependent promotion of VDAC1 oligomerization and suppression of Wnt1/ß-catenin pathway.

GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways.

Olaquindox, a quinoxaline 1,4-dioxide derivative, is widely used as a feed additive in many countries. The potential genotoxicity of olaquindox, hence, is of concern. However, the proper mechanism of toxicity was unclear. The aim of the present study was to investigate the effect of growth arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced DNA damage and cell cycle arrest in HepG2 cells. The results showed that olaquindox could induce reactive oxygen species (ROS)-mediated DNA damage and S-phase arrest, where increases of GADD45a, cyclin A, Cdk 2, p21 and p53 protein expression, decrease of cyclin D1 and the activation of phosphorylation-c-Jun N-terminal kinases (p-JNK), phosphorylation-p38 (p-p38) and phosphorylation-extracellular signal-regulated kinases (p-ERK) were involved. However, GADD45a knockdown cells treated with olaquindox could significantly decrease cell viability, exacerbate DNA damage and increase S-phase arrest, associated with the marked activation of p-JNK, p-p38, but not p-ERK. Furthermore, SP600125 and SB203580 aggravated olaquindox-induced DNA damage and S-phase arrest, suppressed the expression of GADD45a. Taken together, these findings revealed that GADD45a played a protective role in olaquindox treatment and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest. Our findings increase the understanding on the molecular mechanisms of olaquindox.

Curcumin Ameliorates Furazolidone-Induced DNA Damage and Apoptosis in Human Hepatocyte L02 Cells by Inhibiting ROS Production and Mitochondrial Pathway.

Furazolidone (FZD), a synthetic nitrofuran derivative, has been widely used as an antibacterial and antiprotozoal agent. Recently, the potential toxicity of FZD has raised concerns, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on FZD-induced cytotoxicity and the underlying mechanism in human hepatocyte L02 cells. The results showed that curcumin pre-treatment significantly ameliorated FZD-induced oxidative stress, characterized by decreased reactive oxygen species (ROS) and malondialdehyde formation, and increased superoxide dismutase, catalase activities and glutathione contents. In addition, curcumin pre-treatment significantly ameliorated the loss of mitochondrial membrane potential, the activations of caspase-9 and -3, and apoptosis caused by FZD. Alkaline comet assay showed that curcumin markedly reduced FZD-induced DNA damage in a dose-dependent manner. Curcumin pre-treatment consistently and markedly down-regulated the mRNA expression levels of p53, Bax, caspase-9 and -3 and up-regulated the mRNA expression level of Bcl-2. Taken together, these results reveal that curcumin protects against FZD-induced DNA damage and apoptosis by inhibiting oxidative stress and mitochondrial pathway. Our study indicated that curcumin may be a promising combiner with FZD to reduce FZD-related toxicity in clinical applications.

Effect of GADD45a on olaquindox-induced apoptosis in human hepatoma G2 cells: Involvement of mitochondrial dysfunction.

Olaquindox, a quinoxaline 1, 4-dioxide derivative, has been widely used as a feed additive for promoting animal growth in China. The aim of present study was to investigate the effect of grow arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced apoptosis in HepG2 cells. The result showed that olaquindox induced the decrease of cell viability in a dose dependent manner. Compared to the control group, olaquindox treatment at 400 and 800µg/mL increased the expression level of GADD45a protein and reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and subsequently increased the expression of Bax while decreased the expression of Bcl-2, leading to the release of cytochrome c (Cyt c). However, knockdown of GADD45a enhanced olaquindox-induced ROS production, disrupted MMP and subsequently caused Cyt c release, then further increased olaquindox- induced cell apoptosis by increasing the activities of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). In conclusion, the results revealed that GADD45a played a critical role in olaquindox-induced apoptosis in HepG2 cells, which may embrace the regulatory ability on the mitochondrial apoptosis pathway.

Quinocetone triggered ER stress-induced autophagy via ATF6/DAPK1-modulated mAtg9a trafficking.

The present study is undertaken to explore quinocetone-induced autophagy and its possible mechanism. Western blotting and green fluorescence protein (GFP)-LC3 vector transfection were performed to determine the ratio of LC3 conversion and its subcellular localization. Results revealed that the quinocetone induced autophagy in time- and dose-dependent manners. Besides, we tested the expressions of immunoglobulin heavy chain binding protein (BiP) and C/EBP homologous protein (CHOP) and the transcription of BiP, HerpUD, and sec24D by western blotting and RT-PCR, respectively. Results showed that quinocetone also induced endoplasmic reticulum (ER) stress during quinocetone-induced autophagy. Furthermore, we observed the cleavage of ATF6, the phosphorylation of MRLC, and the expression of death-associated protein kinase (DAPK1) by western blotting; the transcription of DAPK1 by RT-PCR; and the subcellular localization of ATF6 and mAtg9 by immunofluorescence. These results suggest that quinocetone stimulates the MRLC-mediated mAtg9 trafficking, which is critical for autophagosome formation, via the ATF6 upregulated expression of DAPK1. Last, we generated ATF6 and DAPK1 stable knockdown HepG2 cell lines and found that the conversion ratios of LC3 were decreased upon the treatment of quinocetone. Together, we propose that quinocetone induces autophagy through ER stress signaling pathway-induced cytoskeleton activation.

P21(Waf1/Cip1) plays a critical role in furazolidone-induced apoptosis in HepG2 cells through influencing the caspase-3 activation and ROS generation.

Furazolidone (FZD), a synthetic nitrofuran with a broad spectrum of antimicrobial activities, has been shown to exhibit marked genotoxity and cytotoxicity in vitro, but the proper mechanism was unclear. P21(Waf1/Cip1) (p21), a cyclin-dependent kinase, is critically involved in cell cycle arrest and apoptosis in response to DNA injury. This study was aimed to explore the role of p21 in FZD-induced apoptosis in HepG2 cells and uncover its possible mechanism. Firstly, we demonstrated that FZD (50 µg/mL) treatment increased the mRNA level of p21 but reduced the protein level of p21 by shortening its half-life. Moreover, the degradation of p21 was associated with the inhibition of PI3K/Akt pathway by FZD. Then, the change of p21 protein expression modulated FZD-induced apoptosis. Overexpression of p21 attenuated FZD-induced caspase-3 activation and ROS generation, eventually reduced apoptosis. Conversely, knockdown of p21 by siRNA enhanced FZD-induced those phenomenon. In addition, the influence of p21 on FZD-induced ROS generation might be associated with Nrf2/HO-1 pathway which was a key regulator in defense response against oxidative stress. In conclusion, these findings demonstrated that p21 plays a critical role in FZD-induced apoptosis in HepG2 cells through influencing the caspase-3 activation and ROS generation.

ML-7 amplifies the quinocetone-induced cell death through akt and MAPK-mediated apoptosis on HepG2 cell line.

The study aims at evaluating the combination of the quinocetone and the ML-7 in preclinical hepatocellular carcinoma models. To this end, the effect of quinocetone and ML-7 on apoptosis induction and signaling pathways was analyzed on HepG2 cell lines. Here, we report that ML-7, in a nontoxic concentration, sensitized the HepG2 cells to quinocetone-induced cytotoxicity. Also, ML-7 profoundly enhances quinocetone-induced apoptosis in HepG2 cell line. Mechanistic investigations revealed that ML-7 and quinocetone act in concert to trigger the cleavage of caspase-8 as well as Bax/Bcl-2 ratio up-regulation and subsequent cleavage of Bid, capsases-9 and -3. Importantly, ML-7 weakened the quinocetone-induced Akt pathway activation, but strengthened the phosphorylation of p-38, ERK and JNK. Further treatment of Akt activator and p-38 inhibitor almost completely abolished the ML-7/quinocetone-induced apoptosis. In contrast, the ERK and JNK inhibitor aggravated the ML-7/quinocetone-induced apoptosis, indicating that the synergism critically depended on p-38 phosphorylation and HepG2 cells provoke Akt, ERK and JNK signaling pathways to against apoptosis. In conclusion, the rational combination of quinocetone and ML-7 presents a promising approach to trigger apoptosis in hepatocellular carcinoma, which warrants further investigation.

Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells.

Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2-16 µg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5 µM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p < 0.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.