Effects of Glycyrrhizae Radix on Repeated Restraint Stress-induced Neurochemical and Behavioral Responses

Glycyrrhizae radix (GR) is an herbal medicine that is commonly used in the East Asia for treating a variety of diseases, including stomach disorders. The objective of the present study was to examine the anti-stress effects of GR on repeated stress-induced alterations of anxiety, learning and memory in rats. Restraint stress was administered for 14 days (2 h/day) to the rats in the Control and GR groups (400 mg/kg/day, PO). Starting on the eighth day, the rats were tested for spatial memory on the Morris water maze test (MW) and for anxiety on the elevated plus maze (EPM). We studied the changes of the expressions of cholineacetyl transferase (ChAT) and tyrosine hydroxylase (TH) in the locus coerleus (LC) using immunohistochemistry. The results showed that the rats treated with GR had significantly reduced stress-induced deficits on their learning and memory on the spatial memory tasks. In addition, the ChAT immunoreactivities were increased. Gor the EPM, treatment with GR increased the time spent in the open arms (p<0.001) as compared to that of the control group. Moreover, GR treatment also normalized the increases of the TH expression in the LC (p<0.001). In conclusion, administration of GR improved spatial learning and memory and reduced stress-induced anxiety. Thus, the present results suggest that GR has the potential to attenuate the behavioral and neurochemical impairments caused by stress.     

Neurochemical and Behavioral Consequences of Ethanol and/or Caffeine Exposure: Effects in Zebrafish and Rodents

Zebrafish are increasingly being utilized to model the behavioral and neurochemical effects of pharmaceuticals and, more recently, pharmaceutical interactions. Zebrafish models of stress establish that both caffeine and ethanol influence anxiety, though few studies have implemented co-administration to assess the interaction of anxiety and reward-seeking. Caffeine exposure in zebrafish is teratogenic, causing developmental abnormalities in the cardiovascular, neuromuscular, and nervous systems of embryos and larvae. Ethanol is also a teratogen and, as an anxiolytic substance, may be able to offset the anxiogenic effects of caffeine. Co-exposure to caffeine and alcohol impacts neuroanatomy and behavior in adolescent animal models, suggesting stimulant substances may moderate the impact of alcohol on neural circuit development. Here, we review the literature describing neuropharmacological and behavioral consequences of caffeine and/or alcohol exposure in the zebrafish model, focusing on neurochemistry, locomotor effects, and behavioral assessments of stress/anxiety as reported in adolescent/juvenile and adult animals. The purpose of this review is twofold: (1) describe the work in zebrafish documenting the effects of ethanol and/or caffeine exposure and (2) compare these zebrafish studies with comparable experiments in rodents. We focus on specific neurochemical pathways (dopamine, serotonin, adenosine, GABA), anxiety-type behaviors (assessed with a novel tank, thigmotaxis, shoaling), and locomotor changes resulting from both individual and co-exposure. We compare findings in zebrafish with those in rodent models, revealing similarities across species and identifying conservation of mechanisms that potentially reinforce co-addiction.     

Age-Related Toxicity in Rat Lungs Following Acute and Repeated Ozone Exposure

Abstract

The evidence is inconclusive as to whether age and gender are important determinants of ozone toxicity We carried out an experiment to investigate the possible age- and gender-related differences in pulmonary toxicity following both acute and repeated exposure to ozone. Male and female rats of various ages (1, 3, 9, and 18 mo) were exposed to 0.8 mg O₃/m³ for 1 day (12 h) or for 7 days (12 h/day) during the dark period. Bronchoalveolar lavage (BAL) and biochemical, histopathological, and immunological techniques were used to determine the permeability, antioxidant capacity, tissue morphology, and extent of inflammation in the lungs. Morphological as well as morphometric results showed age-related differences in the extent of pulmonary lesions after 1 and 7 days of ozone exposure; from the age of 3 mo animals became less susceptible to ozone. Pulmonary antioxidant enzyme capacity in control rats appeared to exhibit an age-related decline starting at 3 mo. However, exposure to ozone resulted in an increase in enzyme activities in rats aged 9 and 18 mo. There was no significant overall age-related effect of ozone. However, a different pattern existed between both sexes in their age-related reaction to ozone exposure. The percentage increase of protein and albumin concentrations in BAL increased after acute ozone exposure, peaking at the age of 1 mo. The lesser increase at the age of 9 and 18 mo suggests a decreasing sensitivity in older rats. The gradual decrease of the net percentage of polymorphonuclear leukocytes (PMNs) in BAL after ozone exposure in male rats with age corroborates this suggestion. Ozone exposure decreased the clearance of Listeria bacteria in lungs. There was no significant difference between the various age groups in the resistance to Listeria infection after ozone exposure. It can therefore be concluded that specific toxicity indices including lung tissue damage, increased permeability, and inflammation point to a more pronounced responsiveness of younger animals to ozone. No gender-related differences in the response to ozone were observed for any of the parameters examined. These data support the view that age is a significant predictor of the pulmonary response to ozone, with younger subjects being more sensitive.     

Effects of Exercise Following Exposure to Perfluoroisobutylene

Abstract

Perfluoroisobutylene (PFIB) is a toxic chemical encountered in industry as a pyrolysis product of tetrafluoroethylene polymers, such as Teflon. Inhalation of toxic doses of PFIB results in varying degrees of pulmonary edema. The purpose of this study was to determine if exhaustive exercise would potentiate the toxic effects of PFIB. One group of treadmill-acclimated rats was exercised to exhaustion following a lhnin whole-body exposure to PFIB (approximately 0.75 × LCt50). A second group was similarly exposed but not exercised. Two other groups of rats were sham exposed; one was exercised while one remained sedentary following the sham exposure. Twenty-four hours after exposure, the animals were sacrificed; the lungs were removed and weighed, and a portion was collected for histopathologic examination. The remaining lung tissue was allowed to dry to constant weight. There was a significant increase in the amount of pulmonary edema and associated pulmonary pathology in rats exercised following exposure to PFIB. Exposure to this concentration of PFIB caused no decrement in immediate postexposure endurance. The degree of pulmonary pathology in all rats exposed to PFIB was profound.     

Short-Term Toxicity of Bitumen Upgrading Products in the Rat Following Repeated Dermal Exposure

Light gas oil (B-LGO), heavy gas oil No. 1 (B-HGOI), and heavygas oil No. 2 (B-HGOII) fractions of bitumen upgrading products(BUPs) were applied on the dorsal skin of rats at 25 mg/kg bw/day(low dose), 100 mg/kg bw/day (intermediate dose), and 400 mg/kgbw/day (high dose) for 4 weeks. Control animals received normalsaline while positive controls received a medium boiling coalliquefaction product (CLP) at 100 and 400 mg/kg bw/day. Reducedfood consumption and growth suppression were observed in malesand females treated with B-HGOI, B-HGOII, and CLP, but onlyin males receiving B-LGO. Increased relative spleen, kidney,and liver weights were observed in animals treated with B-HGOI,B-HGOII, and CLP, but not in control or LGO groups. A dose-relatedincrease in absolute and relative liver weight was most markedin animals receiving B-HGOII where a significant increase wasobserved starting at the low dose, followed by those receivingB-HGOI and CLP. Appearance of pale foci on the splenic capsuleand increases in spleen/body weight ratio were limited to animalsreceiving B-HGOI and B-HGOII. Decreases in hematocrit and RBCand increase in percentage of reticulocytes were observed inanimals of both sexes receiving B-HGOI and B-HGOII. Female ratsappeared to be more severely affected because significant decreasesin hemoglobin and RBC were observed in animals receiving thelow dose of B-HGOII and the intermediate dose of B-HGO-I. Increasedserum cholesterol was observed in B-HGOII-treated females atall dose levels, and in males starting at the intermediate dose.Histological changes were observed in the thymus gland, wheremoderate to marked cortical atrophy was noted in male and femalerats receiving the high dose of B-HGOI and B-HGOII, and in thebone marrow, where the most significant abnormality was thepresence of focal myelofibrosis in some male rats treated withB-HGOI and B-HGOII. Mild to moderate histological changes werefound in the thyroid, liver, and spleen of rats of all treatmentgroups. Changes in the skin included moderate hyperkeratosisin fe males receiving high doses of B-LGO and in animals ofboth sexes receiving high doses of B-HGOI, and moderate to markedepidermal hyperplasia in rats receiving high doses of B-HGOI.Based on these multiple endpoints, the severity of systemictox icity was B-HGOII > B-HGOI > CLP B-LGO. The NOEL wasabout 25 mg/kg bw/day for B-LGO and lower than 25 mg/ kg bw/dayfor B-HGOI and B-HGOII.     

Impulsivity Characterization in the Roman High- and Low-Avoidance Rat Strains: Behavioral and Neurochemical Differences

The selective breeding of Roman high- (RHA) and low-avoidance (RLA) rats for rapid vs extremely poor acquisition of active avoidance behavior in a shuttle-box has generated two phenotypes with different emotional and motivational profiles. The phenotypic traits of the Roman rat lines/strains (outbred or inbred, respectively) include differences in sensation/novelty seeking, anxiety/fearfulness, stress responsivity, and susceptibility to addictive substances. We designed this study to characterize differences between the inbred RHA-I and RLA-I strains in the impulsivity trait by evaluating different aspects of the multifaceted nature of impulsive behaviors using two different models of impulsivity, the delay-discounting task and five-choice serial reaction time (5-CSRT) task. Previously, rats were evaluated on a schedule-induced polydipsia (SIP) task that has been suggested as a model of obsessive-compulsive disorder. RHA-I rats showed an increased acquisition of the SIP task, higher choice impulsivity in the delay-discounting task, and poor inhibitory control as shown by increased premature responses in the 5-CSRT task. Therefore, RHA-I rats manifested an increased impulsivity phenotype compared with RLA-I rats. Moreover, these differences in impulsivity were associated with basal neurochemical differences in striatum and nucleus accumbens monoamines found between the two strains. These findings characterize the Roman rat strains as a valid model for studying the different aspects of impulsive behavior and for analyzing the mechanisms involved in individual predisposition to impulsivity and its related psychopathologies.     

Toxic Effects of Sarin in Rats at Three Months Following Single or Repeated Low‐Level Inhalation Exposure

Abstract: Male albino Wistar rats were once or repeatedly exposed to three various low concentrations of sarin for 60 min. in the inhalation chamber. The clinical status of control as well as sarin‐poisoned rats was tested 3 months after exposure to sarin using biochemical, haematological, neurophysiological, behavioural and immunotoxicological methods. While biochemical and haematological parameters, including the activities of cholinesterases in erythrocytes, plasma and various organs (brain, diaphragm), did not differ from the control values regardless of the sarin concentration used, few signs of sarin‐induced neurotoxicity and immunotoxicity in sarin‐poisoned rats were demonstrated. This was especially true when the single exposure of rats to non‐convulsive symptomatic concentration and repeated exposure of rats to clinically asymptomatic concentration of sarin was used. In rats repeatedly poisoned with clinically asymptomatic concentrations of sarin, the alteration of the gait characterized by ataxia, the increase in the stereotyped behaviour, the increase in the excitability of the central nervous system following the administration of the convulsive drug pentamethylenetetrazol were observed. In rats poisoned with non‐convulsive symptomatic concentration of sarin, the subtle supression of spontaneous, as well as lipopolysaccharides‐stimulated, proliferation of spleen lymphocytes and the bactericidal activity of peritoneal macrophages was primarily observed besides the signs of neurotoxicity. Our findings confirm that both non‐convulsive symptomatic and clinically asymptomatic concentrations of sarin can only cause very few, subtle long‐term signs of neurotoxicity and immunotoxicity in sarin‐poisoned rats when the rats were exposed to asymptomatic sarin concentrations repeatedly.     

Gender Effects Following Repeated Administration of Cocaine and Alcohol in Humans

Meta-analysis is applied to aggregate-level studies that model the demand for cigarettes using static, myopic, or rational addiction frameworks in an attempt to synthesize key findings in the literature and to identify determinants of the variation in reported price elasticity estimates across studies. The results suggest that the rational addiction framework produces statistically similar estimates to the static framework but that studies that use the myopic framework tend to report more elastic price effects. Studies that applied panel data techniques or controlled for cross-border smuggling reported more elastic price elasticity estimates, whereas the use of instrumental variable techniques and time trends or time dummy variables produced less elastic estimates. The finding that myopic models produce different estimates than either of the other two model frameworks underscores that careful attention must be given to time series properties of the data.     

MDMA Elicits Behavioral and Neurochemical Sensitization in Rats

Rats were treated with repeated injections of saline or one of two doses of (±)3,4-methylenedioxymethamphetamine (MDMA; 5 or 20 mg/kg, SC). Rats pretreated with either of the two repeated MDMA treatment regimens demonstrated an augmented increase in motor activity to an injection of MDMA made 12 days after the last repeated injection compared with either the first MDMA injection or MDMA given to animals pretreated with repeated saline. Furthermore, animals pretreated with the highest dose of repeated MDMA revealed a greater behavioral response to cocaine (15 mg/kg, IP). Microdialysis was conducted in the nucleus accumbens and the capacity of MDMA (5 mg/kg, SC) to elevate extracellular dopamine content was augmented in rats pretreated with repeated MDMA compared with the animals pretreated with repeated saline. These data reveal repeated MDMA administration produces behavioral sensitization and enhanced dopamine transmission in the nucleus accumbens of rats.     

Reward Sensitization: Effects of Repeated Nicotine Exposure and Withdrawal in Mice

Tobacco dependence is an addiction with high rates of relapse, resulting in multiple quit attempts in individuals who are trying to stop smoking. How these multiple cycles of smoking and withdrawal contribute to nicotine dependence, long-term alterations in brain reward systems, and nicotine receptor regulation is unknown. Therefore, to evaluate how multiple exposures of nicotine and withdrawal periods modulate rewarding properties of nicotine, we used intracranial self-stimulation to measure alterations in the threshold of brain stimulation reward. In addition, we employed the conditioned place preference (CPP) paradigm to evaluate positive context conditioning following each withdrawal period and measured levels of neuronal nicotinic receptors in cortex, striatum, and hippocampus. We found that repeated nicotine exposure and withdrawal enhanced brain stimulation reward and reward sensitivity to acute injections of nicotine. This increased reward was reflected by enhanced CPP to nicotine. Chronic nicotine is known to up-regulate nAChRs (nicotinic acetylcholine receptors) and we found that this up-regulation was maintained for up to 8 days of withdrawal in the striatum and in the hippocampus, but not in the cortex, of animals exposed to multiple nicotine exposure and withdrawal periods. These results demonstrate that repeated exposures to nicotine, followed by withdrawal, induce a persistent increase in both brain reward function and sensitivity to the hedonic value of nicotine and long-lasting up-regulation of neuronal nicotinic receptors. Together, these data suggest that a continuing increase in brain reward function and enhanced sensitivity to nicotine reward following repeated withdrawal periods may be one reason why smokers relapse frequently.     

Effects of Repeated Cocaine Exposure on Habit Learning and Reversal by N-Acetylcysteine

Exposure to drugs of abuse can result in a loss of control over both drug- and nondrug-related actions by accelerating the transition from goal-directed to habitual control, an effect argued to reflect changes in glutamate homeostasis. Here we examined whether exposure to cocaine accelerates habit learning and used in vitro electrophysiology to investigate its effects on measures of synaptic plasticity in the dorsomedial (DMS) and dorsolateral (DLS) striatum, areas critical for actions and habits, respectively. We then administered N-acetylcysteine (NAC) in an attempt to normalize glutamate homeostasis and hence reverse the cellular and behavioral effects of cocaine exposure. Rats received daily injections of cocaine (30 mg/kg) for 6 days and were then trained to lever press for a food reward. We used outcome devaluation and whole-cell patch-clamp electrophysiology to assess the behavioral and cellular effects of cocaine exposure. We then examined the ability of NAC to reverse the effects of cocaine exposure on these measures. Cocaine treatment produced a deficit in goal-directed action, as assessed by outcome devaluation, and increased the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) in the DMS but not in the DLS. Importantly, NAC treatment both normalized EPSC frequency and promoted goal-directed control in cocaine-treated rats. The promotion of goal-directed control has the potential to improve treatment outcomes in human cocaine addicts.     

Granulomatous Dermatitis in New Zealand White Rabbits Following 9-Day Repeated Cutaneous Exposure to Methyldimethoxysilane

Abstract

Male and female New Zealand White rabbits were exposed to 0.0,0.05,0.1, or 0.2 ml/kg of undiluted methyldimethoxysilane (MDMS), corresponding to 43, 85, or 171 mg/kg, applied under occlusive dressing to the clipped dorsal trunk skin for nine doses over an 11 day period. Five animals/group/gender (10/gender in the 85 mg/kg/day group) were euthanized at the end of the exposure period. Five animals/group/gender from the high-dose and control groups were retained for a 2 week recovery period and then euthanized. The only abnormal findings involved the treated skin. Clinical observations included mild to moderate irritation of the treated skin, affecting mainly high-dose group animals euthanized immediately after the exposure period, with females being slightly more sensitive. Significant gross and microscopic lesions were seen in the treated skin of animals receiving 0.1 ml/kg/day and higher. Gross lesions consisted of erythema, ecchymoses, exfoliation, excoriation, fissures, ulceration, and necrosis. Microscopic lesions included hyperkeratosis, acanthosis, congestion, hemorrhage, epidermitis, dermatitis, and ulceration. Dermal fibrosis and prominent granulomatous inflammation, associated with pigmented granular foreign material, was found in the superficial dermis. After the 2 week recovery period, exfoliation was the only gross skin lesion found in high dose group animals. Microscopic skin lesions consisted of marked granulomatous dermatitis and a fibrotic reaction associated with the foreign material, as well as microscopic examination of the skin of recovery group animals revealed scattered electron-dense deposits in the superficial dermis, which were proven, on elemental analysis, to contain silicon, possibly in the form of a polymer from absorbed MDMS or its breakdown products. There were no abnormal clinical pathologic findings in animals euthanized immediately after exposure, but a significant monocytopenia occurred in high-dose-group male rabbits from the recovery group, which may be due to sequentration of circulating monocytes in the skin at the site of granulomatous inflammation.     

DNA Methylation,Cell Proliferation,and Histopathology in Rats Following Repeated Inhalation Exposure to Dimethyl Sulfate

Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N⁷-Methylguanine and N³-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N⁷-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N⁷-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N⁷-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N³-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at ≥0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.     

The Effects of Astragalus Membranaceus on Repeated Restraint Stress-induced Biochemical and Behavioral Responses

Astragalus Membranaceus (AM) is a useful Korean herb that has been clinically prescribed for stress-related illness. The objective of the present study was to examine the anti-stress effects of AM on repeated stress-induced alterations of anxiety, learning and memory in rats. Restraint stress was administered for 14 days (2h/day) and AM (400mg/kg) given by oral administration, in the AM group, for the same period. Starting on the eighth day, the rats were tested for spatial memory on the Morris water maze test (MW) and for anxiety on the elevated plus maze (EPM). Changes of expression on immunohistochemistry were studied for cholineacetyl transferase (ChAT) and tyrosine hydroxylase (TH) in the brain. The results showed that the rats treated with AM had significantly reduced stress-induced deficits on learning and memory on the spatial memory tasks. In addition, the ChAT immunoreactivities were increased. In the EPM, treatment with AM increased the time spent in the open arms (p<0.001) compared to the control group. In addition, AM treatment also normalized increases of TH expression in the LC (p<0.001). In conclusion, administration of AM improved spatial learning and memory and reduced stress-induced anxiety. Thus, the present results suggest that AM is able to recover behavioral and neurochemical impairments induced by stress.     

Modulation of Muscarinic Receptors and Acetylcholinesterase Activity in Lymphocytes and in Brain Areas Following Repeated Organophosphate Exposure in Rats

Repeated exposures to organophosphorus (OP) insecticides hasbeen shown to cause a decrease of cholinergic muscarinic receptors(mAChR) in brain and in peripheral tissues. These changes arebelieved to be involved in the development of tolerance to OPtoxicity and may play a role in cognitive dysfunctions observedfollowing repeated OP exposure. Recently, mAChRs identifiedin circulating lymphocytes have been shown to be modulated similarlyto brain mAChRs following repeated OP exposure, suggesting thatthese peripheral cells may be useful as indicators of mAChRchanges in the central nervous system. This study was designedto further investigate whether mAChRs on lymphocytes could serveas a biomarker for changes in brain mAChRs during prolongedOP exposure and during recovery from such exposure. Using themAChR antagonist [³H]quinuclidinyl benzilate (QNB) to labelmAChRs, we found that exposure to disulfoton for 14 days (2mg/kg/day by gavage) caused a significant decrease (25–35%)in muscarinic receptors density in the cerebral cortex, hippocampus,and striatum, as well as in circulating lymphocytes. The declineof mAChR density in lymphocytes paralleled those observed inbrain, particularly in cortex and hippocampus, during exposureto disulfoton; however, while brain mAChR levels recovered slowlyalter termination of exposure and remained significantly reduced4 weeks after the last treatment, [³H]QNB binding in lymphocytesrecovered rapidly within 1 week. Similarly, lymphocyte acetylcholinesterase(AChE) activity was significantly inhibited and correlated wellwith brain AChE activity during exposure, but the recovery wasrapid relative to AChE activity in brain. On the other hand,the recovery of red blood cell AChE paralleled that observedin brain tissue. These findings suggest that lymphocytes maybe useful as peripheral markers to monitor for changes in mAChRand AChE in brain tissue during repeated OP exposure, but arenot suitable markers for these CNS parameters alter terminationof exposure.     

The Influence of Sarin on Various Physiological Functions in Rats Following Single or Repeated Low-Level Inhalation Exposure

Long-term effects of low doses of highly toxic organophosphorus agent sarin on various hematological and biochemical markers and physiological functions were studied in rats exposed to sarin by inhalation. The results indicate that low-level sarin-exposed rats show long-term increase in studied markers of stress and decrease in synthesis of DNA de novo without the disturbance of the functions of cholinergic nervous system. Moreover, sarin at low doses is able to induce some neurotoxic effects including an increase in the excitability of central nervous system in rats at 3 mo following inhalation exposure. Relatively long-term spatial discrimination impairments in rats exposed to low-level sarin was demonstrated too. Therefore, nerve agents such as sarin seem to be harmful not only at high, clinically symptomatic doses but also at low doses without acute clinical manifestation of overtimulation of cholinergic nervous system because of long-term manifestation of alteration of neurophysiological and neurobehavioral functions in sarin-exposed rats.     

Enhanced Eryptosis Following Juglone Exposure

Juglone, a quinone isolated from Juglans mandshurica Maxim, has previously been shown to be effective against malignancy. The effect is at least partially due to stimulation of suicidal death or apoptosis of tumour cells. On the other hand, juglone has been shown to counteract apoptosis, for example, of neurons. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca²⁺ activity [(Ca²⁺)_i]. This study explored whether juglone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine exposure at the erythrocyte surface from FITC annexin V binding, ceramide abundance from binding of fluorescent antibodies in flow cytometry and cytosolic ATP with a luciferin–luciferase‐based assay. As a result, a 24‐hr exposure of human erythrocytes to juglone (5 μM) significantly decreased erythrocyte forward scatter. Juglone (1–5 μM) significantly increased the percentage of annexin V binding cells. Juglone (5 μM) significantly increased ceramide abundance at the erythrocyte surface and decreased erythrocyte ATP concentration. The effect of juglone (10 μM) on annexin V binding was slightly but significantly blunted by removal of extracellular Ca²⁺ and by addition of protein kinase C (PKC) inhibitor staurosporine (1 μM). In conclusion, juglone stimulates suicidal erythrocyte death or eryptosis at least in part by upregulation of ceramide abundance, energy depletion and activation of PKC.