Hepatic pyruvate and alanine metabolism are critical and complementary for maintenance of antioxidant capacity and resistance to oxidative insult.

OBJECTIVE: Mitochondrial pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, and NADH production. As a result, the mitochondrial pyruvate carrier (MPC) complex has emerged as a promising therapeutic target in metabolic diseases. Clinical trials are currently underway. However, recent in vitro data indicate that MPC inhibition diverts glutamine/glutamate away from glutathione synthesis and toward glutaminolysis to compensate for loss of pyruvate oxidation, possibly sensitizing cells to oxidative insult. Here, we explored this in vivo using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress. METHODS: We used pharmacological and genetic approaches to inhibit MPC2 and alanine aminotransferase 2 (ALT2), individually and concomitantly, in mice and cell culture models and determined the effects on APAP hepatotoxicity. RESULTS: We found that MPC inhibition sensitizes the liver to APAP-induced injury in vivo only with concomitant loss of alanine aminotransferase 2 (ALT2). Pharmacological and genetic manipulation of neither MPC2 nor ALT2 alone affected APAP toxicity, but liver-specific double knockout (DKO) significantly worsened APAP-induced liver damage. Further investigation indicated that DKO impaired glutathione synthesis and increased urea cycle flux, consistent with increased glutaminolysis, and these results were reproducible in vitro. Finally, induction of ALT2 and post-treatment with dichloroacetate both reduced APAP-induced liver injury, suggesting new therapeutic avenues. CONCLUSIONS: Increased susceptibility to APAP toxicity requires loss of both the MPC and ALT2 in vivo, indicating that MPC inhibition alone is insufficient to disrupt redox balance. Furthermore, the results from ALT2 induction and dichloroacetate in the APAP model suggest new metabolic approaches to the treatment of liver damage.

Factors associated with ever using cannabidiol in a cohort of younger pregnant people.

One in four Americans have used cannabidiol (CBD) products in the past year, and use has become prevalent in many Western countries with recent deregulation from a controlled or illicit substance to an unrestricted product. CBD is also marketed to pregnant people to treat common medical conditions. However, preclinical work has linked cannabidiol exposure to embryotoxicity, as well as neuroendocrine, reproductive, and behavioral effects in offspring. No studies have examined the prevalence or correlates of CBD use among pregnant people. Demographic, medical, and psychosocial correlates of cannabidiol use were examined in the YoungMoms study, a cohort of pregnant people under the age of 22, a population that is at high risk for cannabis use during pregnancy. Few of the participants (n = 186; 75% Black or Biracial) reported use of cannabidiol during pregnancy, but one in five had tried these products. Participants who reported ever using CBD were more likely to report alcohol and other drug use prior to pregnancy, controlling for race.As the use of CBD among people of reproductive age is increasingly prevalent, more research on CBD use in pregnant human populations is needed to investigate the effects of CBD on fetal development and infant outcomes.

Proteomics Indicates Lactate Dehydrogenase Is Prognostic in Acetaminophen-Induced Acute Liver Failure Patients and Reveals Altered Signaling Pathways.

Better biomarkers to predict death early in acute liver failure (ALF) are needed. To that end, we obtained early (study day 1) and later (day 3) serum samples from transplant-free survivors (n = 28) and nonsurvivors (n = 30) of acetaminophen-induced ALF from the NIH-sponsored Acute Liver Failure Study Group and from control volunteers (n = 10). To identify proteins that increase early in serum during ALF, we selected individuals from this cohort for whom alanine aminotransferase was lower on day 1 than day 3, indicating a time point before peak injury (n = 10/group). We then performed untargeted proteomics on their day 1 samples. Out of 1682 quantifiable proteins, 361 were ≥ 4-fold elevated or decreased in ALF patients versus controls and 16 of those were further elevated or decreased ≥ 4-fold in nonsurvivors versus survivors, indicating potential to predict death. Interestingly, 1 of the biomarkers was lactate dehydrogenase (LDH), which is already measured in most clinical laboratories. To validate our proteomics results and to confirm the prognostic potential of LDH, we measured LDH activity in all day 1 and 3 samples from all 58 ALF patients. LDH was elevated in the nonsurvivors versus survivors on both days. In addition, it had prognostic value similar to the model for end-stage liver disease and outperformed the King's College Criteria, while a combination of model for end-stage liver disease and LDH together outperformed either alone. Finally, bioinformatics analysis of our proteomics data revealed alteration of numerous signaling pathways that may be important in liver regeneration. Overall, we conclude LDH can predict death in APAP-induced ALF.

Short-Term Safety of Repeated Acetaminophen Use in Patients With Compensated Cirrhosis.

Current guidelines recommend restricting acetaminophen (APAP) use in patients with cirrhosis, but evidence to support that recommendation is lacking. Prior studies focused on pharmacokinetics (PK) of APAP in cirrhosis but did not rigorously examine clinical outcomes, sensitive biomarkers of liver damage, or serum APAP-protein adducts, which are a specific marker of toxic bioactivation. Hence, the goal of this pilot study was to test the effects of regularly scheduled APAP dosing in a well-defined compensated cirrhosis group compared to control subjects without cirrhosis, using the abovementioned outcomes. After a 2-week washout, 12 subjects with and 12 subjects without cirrhosis received 650 mg APAP twice per day (1.3 g/day) for 4 days, followed by 650 mg on the morning of day 5. Patients were assessed in-person at study initiation (day 1) and on days 3 and 5. APAP-protein adducts and both conventional (alanine aminotransferase) and sensitive (glutamate dehydrogenase [GLDH], full-length keratin 18 [K18], and total high-mobility group box 1 protein) biomarkers of liver injury were measured in serum on the mornings of days 1, 3, and 5, with detailed PK analysis of APAP, metabolites, and APAP-protein adducts throughout day 5. No subject experienced adverse clinical outcomes. GLDH and K18 were significantly different at baseline but did not change in either group during APAP administration. In contrast, clearance of APAP-protein adducts was dramatically delayed in the cirrhosis group. Minor differences for other APAP metabolites were also detected. Conclusion: Short-term administration of low-dose APAP (650 mg twice per day, <1 week) is likely safe in patients with compensated cirrhosis. These data provide a foundation for future studies to test higher doses, longer treatment, and subjects who are decompensated, especially in light of the remarkably delayed adduct clearance in subjects with cirrhosis.

Exogenous phosphatidic acid reduces acetaminophen-induced liver injury in mice by activating hepatic interleukin-6 signaling through inter-organ crosstalk.

We previously demonstrated that endogenous phosphatidic acid (PA) promotes liver regeneration after acetaminophen (APAP) hepatotoxicity. Here, we hypothesized that exogenous PA is also beneficial. To test that, we treated mice with a toxic APAP dose at 0 h, followed by PA or vehicle (Veh) post-treatment. We then collected blood and liver at 6, 24, and 52 h. Post-treatment with PA 2 h after APAP protected against liver injury at 6 h, and the combination of PA and N-acetyl-l-cysteine (NAC) reduced injury more than NAC alone. Interestingly, PA did not affect canonical mechanisms of APAP toxicity. Instead, transcriptomics revealed that PA activated interleukin-6 (IL-6) signaling in the liver. Consistent with that, serum IL-6 and hepatic signal transducer and activator of transcription 3 (Stat3) phosphorylation increased in PA-treated mice. Furthermore, PA failed to protect against APAP in IL-6-deficient animals. Interestingly, IL-6 expression increased 18-fold in adipose tissue after PA, indicating that adipose is a source of PA-induced circulating IL-6. Surprisingly, however, exogenous PA did not alter regeneration, despite the importance of endogenous PA in liver repair, possibly due to its short half-life. These data demonstrate that exogenous PA is also beneficial in APAP toxicity and reinforce the protective effects of IL-6 in this model.

Safety and Molecular-Toxicological Implications of Cannabidiol-Rich Cannabis Extract and Methylsulfonylmethane Co-Administration.

Cannabidiol (CBD) is a biologically active, non-psychotropic component of Cannabis sativa whose popularity has grown exponentially in recent years. Besides a wealth of potential health benefits, ingestion of CBD poses risks for a number of side effects, of which hepatotoxicity and CBD/herb-drug interactions are of particular concern. Here, we investigated the interaction potential between the cannabidiol-rich cannabis extract (CRCE) and methylsulfonylmethane (MSM), a popular dietary supplement, in the mouse model. For this purpose, 8-week-old male C57BL6/J mice received MSM-containing water (80 mg/100 mL) ad libitum for 17 days. During the last three days of treatment, mice received three doses of CRCE administered in sesame oil via oral gavage (123 mg/kg/day). Administration of MSM alone did not result in any evidence of liver toxicity and did not induce expression of mouse cytochrome P450 (CYP) enzymes. Administration of CRCE did produce significant (p < 0.05) increases in Cyp1a2, Cyp2b10, Cyp2c29, Cyp3a4, Cyp3a11, Cyp2c65, and Cyp2c66 messenger RNA, however, this effect was not amplified by MSM/CRCE co-treatment. Similarly, no evidence of liver toxicity was observed in MSM/CRCE dosed mice. In conclusion, short-term MSM/CRCE co-administration did not demonstrate any evidence of hepatotoxicity in the mouse model.

Pre-treatment twice with liposomal clodronate protects against acetaminophen hepatotoxicity through a pre-conditioning effect.

BACKGROUND AND AIM: Acetaminophen (APAP) overdose is a major cause of acute liver injury, but the role of macrophages in propagation of the hepatotoxicity is controversial. Early research revealed that macrophage inhibitors protect against APAP injury. However, later work demonstrated that macrophage ablation by acute pre-treatment with liposomal clodronate (LC) exacerbates the toxicity. To our surprise, during other studies, we observed that pre-treatment twice with LC seemed to protect against APAP hepatotoxicity, in contrast to acute pre-treatment. The aim of this study was to confirm that observation and to explore the mechanisms. METHODS: We treated mice with empty liposomes (LE) or LC twice per week for 1 week before APAP overdose and collected blood and liver tissue at 0, 2, and 6 h post-APAP. We then measured liver injury (serum ALT activity, histology), APAP bioactivation (total glutathione, APAP-protein adducts), oxidative stress (oxidized glutathione [GSSG]), glutamate cysteine-ligase subunit c (Gclc) mRNA, and nuclear factor erythroid 2-related factor (Nrf2) immunofluorescence. We also confirmed ablation of macrophages by F4/80 immunohistochemistry. RESULTS: Pre-treatment twice with LC dramatically reduced F4/80 staining, protected against liver injury, and reduced oxidative stress at 6 h post-APAP, without affecting APAP bioactivation. Importantly, Gclc mRNA was higher in the LC group at 0 h and total glutathione was higher at 2 h, indicating accelerated glutathione re-synthesis after APAP overdose due to greater basal glutamate-cysteine ligase. Oxidative stress was lower in the LC groups at both time points. Finally, total Nrf2 immunofluorescence was higher in the LC group. CONCLUSIONS: We conclude that multiple pre-treatments with LC protect against APAP by accelerating glutathione re-synthesis through glutamate-cysteine ligase. Investigators using two or possibly more LC pre-treatments to deplete macrophages, including peritoneal macrophages, should be aware of this possible confounder.

Identification of Serum Biomarkers to Distinguish Hazardous and Benign Aminotransferase Elevations.

The standard circulating biomarker of liver injury in both clinical settings and drug safety testing is alanine aminotransferase (ALT). However, ALT elevations sometimes lack specificity for tissue damage. To identify novel serum biomarkers with greater specificity for injury, we combined unique animal models with untargeted proteomics, followed by confirmation with immunoblotting. Using proteomics, we identified 109 proteins in serum from mice with acetaminophen (APAP)-induced liver injury that were not detectable in serum from mice with benign ALT elevations due to high-dose dexamethasone (Dex). We selected 4 (alcohol dehydrogenase 1A1 [Aldh1a1], aldehyde dehydrogenase 1 [Adh1], argininosuccinate synthetase 1 [Ass1], and adenosylhomocysteinase [Ahcy]) with high levels for further evaluation. Importantly, all 4 were specific for injury when using immunoblots to compare serum from Dex-treated mice and mice with similar lower ALT elevations due to milder models of APAP or bromobenzene-induced liver injury. Immunoblotting for ALDH1A1, ADH1, and ASS1 in serum from APAP overdose patients without liver injury and APAP overdose patients with mild liver injury revealed that these candidate biomarkers can be detected in humans with moderate liver injury as well. Interestingly, further experiments with serum from rats with bile duct ligation-induced liver disease indicated that Aldh1a1 and Adh1 are not detectable in serum in cholestasis and may therefore be specific for hepatocellular injury and possibly even drug-induced liver injury, in particular. Overall, our results strongly indicate that ALDH1A1, ADH1, and ASS1 are promising specific biomarkers for liver injury. Adoption of these biomarkers could improve preapproval drug safety assessment.

Paradoxical Patterns of Sinusoidal Obstruction Syndrome-Like Liver Injury in Aged Female CD-1 Mice Triggered by Cannabidiol-Rich Cannabis Extract and Acetaminophen Co-Administration.

The goal of this study was to investigate the potential for a cannabidiol-rich cannabis extract (CRCE) to interact with the most common over-the-counter drug and the major known cause of drug-induced liver injury-acetaminophen (APAP)-in aged female CD-1 mice. Gavaging mice with 116 mg/kg of cannabidiol (CBD) [mouse equivalent dose (MED) of 10 mg/kg of CBD] in CRCE delivered with sesame oil for three consecutive days followed by intraperitoneally (i.p.) acetaminophen (APAP) administration (400 mg/kg) on day 4 resulted in overt toxicity with 37.5% mortality. No mortality was observed in mice treated with 290 mg/kg of CBD+APAP (MED of 25 mg/kg of CBD) or APAP alone. Following CRCE/APAP co-administration, microscopic examination revealed a sinusoidal obstruction syndrome-like liver injury-the severity of which correlated with the degree of alterations in physiological and clinical biochemistry end points. Mechanistically, glutathione depletion and oxidative stress were observed between the APAP-only and co-administration groups, but co-administration resulted in much greater activation of c-Jun N-terminal kinase (JNK). Strikingly, these effects were not observed in mice gavaged with 290 mg/kg CBD in CRCE followed by APAP administration. These findings highlight the potential for CBD/drug interactions, and reveal an interesting paradoxical effect of CBD/APAP-induced hepatotoxicity.

Hepatotoxicity of a Cannabidiol-Rich Cannabis Extract in the Mouse Model.

The goal of this study was to investigate Cannabidiol (CBD) hepatotoxicity in 8-week-old male B6C3F1 mice. Animals were gavaged with either 0, 246, 738, or 2460 mg/kg of CBD (acute toxicity, 24 h) or with daily doses of 0, 61.5, 184.5, or 615 mg/kg for 10 days (sub-acute toxicity). These doses were the allometrically scaled mouse equivalent doses (MED) of the maximum recommended human maintenance dose of CBD in EPIDIOLEX® (20 mg/kg). In the acute study, significant increases in liver-to-body weight (LBW) ratios, plasma ALT, AST, and total bilirubin were observed for the 2460 mg/kg dose. In the sub-acute study, 75% of mice gavaged with 615 mg/kg developed a moribund condition between days three and four. As in the acute phase, 615 mg/kg CBD increased LBW ratios, ALT, AST, and total bilirubin. Hepatotoxicity gene expression arrays revealed that CBD differentially regulated more than 50 genes, many of which were linked to oxidative stress responses, lipid metabolism pathways and drug metabolizing enzymes. In conclusion, CBD exhibited clear signs of hepatotoxicity, possibly of a cholestatic nature. The involvement of numerous pathways associated with lipid and xenobiotic metabolism raises serious concerns about potential drug interactions as well as the safety of CBD.

Expression of drug metabolizing enzymes and transporters in the cochlea: Implications for drug delivery and ototoxicity.

Inner ear drug delivery is a major area of research and development, but relatively little is known about basic drug metabolism in the cochlea. Additionally, the use of potentially ototoxic drugs such as NSAIDs, chemotherapeutics and aminoglycosides is common, but little is known about the role of metabolism in ototoxicity of those drugs. To address those issues, we compared expression of major Cytochromes P450 (Cyps), UDP-glucuronosyl-transferases (Ugts), sulfotransferases (Sults), and drug transporters between cochleae and liver, an organ with high expression, in mice using qPCR and enzyme kinetics. Together, the tested drug-metabolizing enzymes (DMEs) and transporters account for metabolism of approximately 70-80% of all medically important drugs in the body. Expression of most Cyps was low in the cochlea compared to liver, but three displayed similar expression levels to the liver, and one (Cyp2c65) had significantly higher levels of expression in the cochlea (1.9 ±â€¯0.06 fold vs. liver). Enzyme kinetics revealed undetectable levels of p450 activity in the cochlea, especially as compared to the liver. Similar results were obtained for expression of Ugts and Sults. Interestingly, expression of most transporters was also low, with one major exception: Mdr1/P-glycoprotein (P-gp), which is generally thought to be highly expressed in liver and poorly expressed in most of the nervous system, was 3-fold greater in cochlea. Importantly, P-gp is known to protect other tissues from toxicity of cancer drugs by acting as an efflux pump. Our data demonstrate overall low levels of expression of DMEs and transporters in the cochlea, and identify a few that may be important to consider when designing and testing drugs for local delivery to the inner ear.

Decaffeinated Green Tea Extract Does Not Elicit Hepatotoxic Effects and Modulates the Gut Microbiome in Lean B6C3F1 Mice.

The main purpose of this study was to investigate the hepatotoxic potential and effects on the gut microbiome of decaffeinated green tea extract (dGTE) in lean B6C3F1 mice. Gavaging dGTE over a range of 1X-10X mouse equivalent doses (MED) for up to two weeks did not elicit significant histomorphological, physiological, biochemical or molecular alterations in mouse livers. At the same time, administration of dGTE at MED comparable to those consumed by humans resulted in significant modulation of gut microflora, with increases in Akkermansia sp. being most pronounced. Results of this study demonstrate that administration of relevant-to-human-consumption MED of dGTE to non-fasting mice does not lead to hepatotoxicity. Furthermore, dGTE administered to lean mice, caused changes in gut microflora comparable to those observed in obese mice. This study provides further insight into the previously reported weight management properties of dGTE; however, future studies are needed to fully evaluate and understand this effect.

The inhibitor of glycerol 3-phosphate acyltransferase FSG67 blunts liver regeneration after acetaminophen overdose by altering GSK3ß and Wnt/ß-catenin signaling.

Repair mechanisms after acetaminophen (APAP) hepatotoxicity are poorly understood. We recently discovered that phosphatidic acid (PA) increases in mice and humans after APAP overdose, and is critical for liver regeneration. Here, we hypothesized that PA inhibits glycogen synthase kinase-3ß (GSK3ß), a component of canonical Wnt/ß-catenin signaling, after APAP overdose. To test that, we treated mice with 300 mg/kg APAP at 0 h followed by vehicle or 20 mg/kg of the glycerol 3-phosphate acyltransferase inhibitor FSG67 at 3, 24 and 48 h. Some mice also received the GSK3 inhibitor L803-mts. Blood and liver were collected at multiple time points. Consistent with our earlier results, FSG67 did not affect toxicity (ALT, histology), APAP bioactivation (total glutathione), or oxidative stress (oxidized glutathione), but did reduce expression of proliferating cell nuclear antigen (PCNA) at 52 h. We then measured GSK3ß phosphorylation and found it was dramatically decreased by FSG67 at 24 h, before PCNA dropped. Expression of cyclin D1, downstream of Wnt/ß-catenin, was also reduced. To determine if the effect of FSG67 on GSK3ß is important, we treated mice with FSG67 and L803-mts after APAP. Importantly, L803-mts rescued hepatocyte proliferation and survival. Our data indicate PA and lysoPA may support recovery after APAP overdose by inhibiting GSK3ß.

Use of a Video Scoring Anchor for Rapid Serial Assessment of Social Communication in Toddlers.

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency-which is foundational for both social learning and social competency-is hypothesized to be disrupted in autism spectrum disorder (ASD). The ability to quantify the full range of RSB during the toddler period, when core symptoms of ASD often arise, is pivotal for evaluating early risk for ASD, characterizing social development, and tracking response to early interventions. However, important parameters of variation in RSB-especially prior to the development of verbal language-can be nuanced and difficult to characterize using questionnaire-based methods. To address this challenge, we developed a system for measuring quantitative variation in RSB in toddlers (ages 18 - 30 months) that incorporated not only standard questionnaire data from caregivers but also a novel set of video-referenced items, through which a respondent compares the behavior of a subject to that observed in a short video of a young child manifesting a highly competent level of social communication. Testing of this measure in a general population sample of twins confirmed that both the video-referenced items and the RSB Total Score (video-referenced items plus non-video-referenced items) displayed unimodal, continuous distributions, strong internal consistency, marked preservation of individual differences, and extremely high heritability. In addition, video-referenced items were particularly sensitive to quantifying incremental changes in social communication, a major element of RSB, over the course of early childhood development. Scores on the vrRSB clearly differentiated children with and without ASD and these data comprise an initial validation of this promising method for quantifying early RSB-cross-sectionally, over time, and as a function of early intervention.

Extrahepatic toxicity of acetaminophen: critical evaluation of the evidence and proposed mechanisms.

Research on acetaminophen (APAP) toxicity over the last several decades has focused on the pathophysiology of liver injury, but increasingly attention is paid to other known and possible adverse effects. It has been known for decades that APAP causes acute kidney injury, but confusion exists regarding prevalence, and the mechanisms have not been well investigated. More recently, evidence for pulmonary, endocrine, neurological, and neurodevelopmental toxicity has been reported in a number of published experimental, clinical, and epidemiological studies, but the quality of those studies has varied. It is important to view those data critically due to implications for regulation and clinical practice. Here, we review evidence and proposed mechanisms for extrahepatic adverse effects of APAP and weigh weaknesses and strengths in the available data. RELEVANCE FOR PATIENTS: APAP is one of the most commonly used drugs in the West. Although it is generally considered safe when used according to manufacturer recommendations, it has been known for decades that overdose can cause liver injury. Recent studies have suggested that APAP can damage cells in other organs as well, leading to calls for more and stricter regulations, which would limit use of this otherwise effective drug. It is especially important to view claims of developmental effects of antenatal APAP exposure with a critical eye because APAP is currently the only over-the-counter medication recommended for pregnant women to self-treat pain and fever.

Infant viewing of social scenes is under genetic control and is atypical in autism.

Long before infants reach, crawl or walk, they explore the world by looking: they look to learn and to engage, giving preferential attention to social stimuli, including faces, face-like stimuli and biological motion. This capacity-social visual engagement-shapes typical infant development from birth and is pathognomonically impaired in children affected by autism. Here we show that variation in viewing of social scenes, including levels of preferential attention and the timing, direction and targeting of individual eye movements, is strongly influenced by genetic factors, with effects directly traceable to the active seeking of social information. In a series of eye-tracking experiments conducted with 338 toddlers, including 166 epidemiologically ascertained twins (enrolled by representative sampling from the general population), 88 non-twins with autism and 84 singleton controls, we find high monozygotic twin-twin concordance (0.91) and relatively low dizygotic concordance (0.35). Moreover, the characteristics that are the most highly heritable, preferential attention to eye and mouth regions of the face, are also those that are differentially decreased in children with autism (χ2 = 64.03, P < 0.0001). These results implicate social visual engagement as a neurodevelopmental endophenotype not only for autism, but also for population-wide variation in social-information seeking. In addition, these results reveal a means of human biological niche construction, with phenotypic differences emerging from the interaction of individual genotypes with early life experience.

Hearing, reactive metabolite formation, and oxidative stress in cochleae after a single acute overdose of acetaminophen: an in vivo study.

CONTEXT: Although the liver is the primary target organ in acetaminophen (APAP) toxicity, other organs are affected. Previous data suggested that chronic APAP abuse can be ototoxic and the mechanism involves APAP-induced oxidative stress and reactive metabolite (N-acetyl-p-benzoquinone imine, NAPQI)-induced endoplasmic reticulum stress. However, the effect of a single acute overdose on hearing has not been tested. OBJECTIVES: To determine if a single acute APAP overdose causes hearing damage, and to explore possible mechanisms of APAP ototoxicity. MATERIALS AND METHODS: Male C57BL/6 J mice were treated with a single human-relevant overdose of APAP (300 mg APAP per kg bodyweight). Blood, liver and cochleae were harvested at 0, 2, 6 and 24 h post-APAP. In some mice, auditory brainstem responses (ABRs) to a range of frequencies were measured at 24 h. The furosemide plus kanamycin (FS/K) model of drug ototoxicity was used as a positive control for hearing loss. NAPQI formation after APAP was assessed by measuring glutathione depletion and covalent protein binding, and oxidative stress was assessed by measuring glutathione disulfide. RESULTS: There was no evidence of reactive metabolite formation or hearing loss after a single overdose of APAP at a clinically relevant dose. However, there was a transient increase in oxidative stress. DISCUSSION: Although a single acute overdose was not ototoxic, there was evidence of oxidative stress which may support a role for oxidative stress in hearing loss due to chronic APAP abuse. CONCLUSION: A single human-relevant acute overdose of APAP causes transient oxidative stress in cochleae but not hearing loss.

Hair cell counts in a rat model of sound damage: Effects of tissue preparation & identification of regions of hair cell loss.

Exposure to intense sound can damage or kill cochlear hair cells (HC). This loss of input typically manifests as noise induced hearing loss, but it can also be involved in the initiation of other auditory disorders such as tinnitus or hyperacusis. In this study we quantify changes in HC number following exposure to one of four sound damage paradigms. We exposed adult, anesthetized Long-Evans rats to a unilateral 16 kHz pure tone that varied in intensity (114 dB or 118 dB) and duration (1, 2, or 4 h) and sacrificed animals 2-4 weeks later. We compared two different methods of tissue preparation, plastic embedding/sectioning and whole mount dissection, for quantifying hair cell loss as a function of frequency. We found that the two methods of tissue preparation produced largely comparable cochleograms, with whole mount dissections allowing a more rapid evaluation of hair cell number. Both inner and outer hair cell loss was observed throughout the length of the cochlea irrespective of sound damage paradigm. Inner HC loss was either equal to or greater than outer HC loss. Increasing the duration of sound exposures resulted in more severe HC loss, which included all HC lesions observed in an analogous shorter duration exposure.