Parallel evaluation of alternative skin barrier models and excised human skin for dermal absorption studies in vitro.

Skin permeation is a primary consideration in the safety assessment of cosmetic ingredients, topical drugs, and human users handling veterinary medicinal products. While excised human skin (EHS) remains the 'gold standard' for in vitro permeation testing (IVPT) studies, unreliable supply and high cost motivate the search for alternative skin barrier models. In this study, a standardized dermal absorption testing protocol was developed to evaluate the suitability of alternative skin barrier models to predict skin absorption in humans. Under this protocol, side-by-side assessments of a commercially available reconstructed human epidermis (RhE) model (EpiDerm-200-X, MatTek), a synthetic barrier membrane (Strat-M, Sigma-Aldrich), and EHS were performed. The skin barrier models were mounted on Franz diffusion cells and the permeation of caffeine, salicylic acid, and testosterone was quantified. Transepidermal water loss (TEWL) and histology of the biological models were also compared. EpiDerm-200-X exhibited native human epidermis-like morphology, including a characteristic stratum corneum, but had an elevated TEWL as compared to EHS. The mean 6 h cumulative permeation of a finite dose (6 nmol/cm2) of caffeine and testosterone was highest in EpiDerm-200-X, followed by EHS and Strat-M. Salicylic acid permeated most in EHS, followed by EpiDerm-200-X and Strat-M. Overall, evaluating novel alternative skin barrier models in the manner outlined herein has the potential to reduce the time from basic science discovery to regulatory impact.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

Doxorubicin-induced delayed-onset subclinical cardiotoxicity in mice.

Subclinical cardiotoxicity at low total cumulative doxorubicin (DOX) doses can manifest into cardiomyopathy in long-term cancer survivors. However, the underlying mechanisms are poorly understood. In male B6C3F1 mice, assessment of cardiac function by echocardiography was performed at 1, 4, 10, 17, and 24 weeks after exposure to 6, 9, 12, and 24 mg/kg total cumulative DOX doses or saline (SAL) to monitor development of delayed-onset cardiotoxicity. The 6- or 9-mg/kg total cumulative doses resulted in a significant time-dependent decline in systolic function (left ventricular ejection fraction (LVEF) and fractional shortening (FS)) during the 24-week recovery although there was not a significant alteration in % LVEF or % FS at any specific time point during the recovery. A significant decline in systolic function was elicited by the cardiotoxic cumulative DOX dose (24 mg/kg) during the 4- to 24-week period after treatment compared to SAL-treated counterparts. At 24 weeks after DOX treatment, a significant dose-related decrease in the expression of genes and proteins involved in sarcoplasmic reticulum (SR) calcium homeostasis (Ryr2 and Serca2) was associated with a dose-related increase in the transcript level of Casp12 (SR-specific apoptosis) in hearts. These mice also showed enhanced apoptotic activity in hearts indicated by a significant dose-related elevation in the number of apoptotic cardiomyocytes compared to SAL-treated counterparts. These findings collectively suggest that a steady decline in SR calcium handling and apoptosis might be involved in the development of subclinical cardiotoxicity that can evolve into irreversible cardiomyopathy later in life.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Subchronic toxicity evaluation of glucosamine and glucosamine in combination with chondroitin sulfate in obese Zucker rats.

D-glucosamine is a widely consumed dietary supplement used to promote joint health and treat osteoarthritis. It also stimulates intracellular hexosamine flux and increases transforming growth factor ß1 (TGFß1) mRNA expression and insulin resistance in animal studies. The effects of D-glucosamine exposure were investigated in obese Zucker rats. Male (leprfa/leprfa) Zucker rats were exposed to 30, 120, 300 and 600 mg D-glucosamine HCl per kg/day either alone or with chondroitin sulfate (24, 96, 240 and 480 mg/kg/day respectively) for 90 days. After 4 weeks exposure, these doses produced CmaxD-glucosamine concentrations of up to 24 µM in tail vein serum concurrent with a transient 30% increase in blood glucose concentration in the 600 mg/kg/day dose group. D-Glucosamine did not significantly alter body weight, blood glucose or serum insulin levels at any dose tested after 13 weeks exposure, but did increase urinary TGFß1 concentrations. The Zucker rats developed nephropathy and scrotal sores that were related to their hyperglycemia and obesity, and D-glucosamine exposure exacerbated these conditions to a small extent. The incidence of pulmonary osseous metaplasia was increased in rats exposed to D-glucosamine and a single incidence of adrenal osseous metaplasia was noted in one animal exposed to 600/480 mg D-glucosamine HCl/chondroitin sulfate. These lesions may have been treatment related. These studies suggest that the risk of adverse effects of oral D-glucosamine is small compared to that of hyperglycemia in these animals, but the potential for TGFß1-mediated pathologies, such as osseous metaplasia and renal nephropathy may be increased.

Sex-related differential susceptibility to doxorubicin-induced cardiotoxicity in B6C3F1 mice.

Sex is a risk factor for development of cardiotoxicity, induced by the anti-cancer drug, doxorubicin (DOX), in humans. To explore potential mechanisms underlying differential susceptibility to DOX between sexes, 8-week old male and female B6C3F1 mice were dosed with 3mg/kg body weight DOX or an equivalent volume of saline via tail vein once a week for 6, 7, 8, and 9 consecutive weeks, resulting in 18, 21, 24, and 27mg/kg cumulative DOX doses, respectively. At necropsy, one week after each consecutive final dose, the extent of myocardial injury was greater in male mice compared to females as indicated by higher plasma concentrations of cardiac troponin T at all cumulative DOX doses with statistically significant differences between sexes at the 21 and 24mg/kg cumulative doses. A greater susceptibility to DOX in male mice was further confirmed by the presence of cytoplasmic vacuolization in cardiomyocytes, with left atrium being more vulnerable to DOX cardiotoxicity. The number of TUNEL-positive cardiomyocytes was mostly higher in DOX-treated male mice compared to female counterparts, showing a statistically significant sex-related difference only in left atrium at 21mg/kg cumulative dose. DOX-treated male mice also had an increased number of γ-H2A.X-positive (measure of DNA double-strand breaks) cardiomyocytes compared to female counterparts with a significant sex effect in the ventricle at 27mg/kg cumulative dose and right atrium at 21 and 27mg/kg cumulative doses. This newly established mouse model provides a means to identify biomarkers and access potential mechanisms underlying sex-related differences in DOX-induced cardiotoxicity.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

Differential Effects of Silver Nanoparticles and Silver Ions on Tissue Accumulation, Distribution, and Toxicity in the Sprague Dawley Rat Following Daily Oral Gavage Administration for 13 Weeks.

There are concerns within the regulatory and research communities regarding the health impact associated with consumer exposure to silver nanoparticles (AgNPs). This study evaluated particulate and ionic forms of silver and particle size for differences in silver accumulation, distribution, morphology, and toxicity when administered daily by oral gavage to Sprague Dawley rats for 13 weeks. Test materials and dose formulations were characterized by transmission electron microscopy (TEM), dynamic light scattering, and inductively coupled mass spectrometry (ICP-MS). Seven-week-old rats (10 rats per sex per group) were randomly assigned to treatments: AgNP (10, 75, and 110 nm) at 9, 18, and 36 mg/kg body weight (bw); silver acetate (AgOAc) at 100, 200, and 400 mg/kg bw; and controls (2 mM sodium citrate (CIT) or water). At termination, complete necropsies were conducted, histopathology, hematology, serum chemistry, micronuclei, and reproductive system analyses were performed, and silver accumulations and distributions were determined. Rats exposed to AgNP did not show significant changes in body weights or intakes of feed and water relative to controls, and blood, reproductive system, and genetic tests were similar to controls. Differences in the distributional pattern and morphology of silver deposits were observed by TEM: AgNP appeared predominantly within cells, while AgOAc had an affinity for extracellular membranes. Significant dose-dependent and AgNP size-dependent accumulations were detected in tissues by ICP-MS. In addition, sex differences in silver accumulations were noted for a number of tissues and organs, with accumulations being significantly higher in female rats, especially in the kidney, liver, jejunum, and colon.

In vivo genotoxicity of estragole in male F344 rats.

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio-activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven-week-old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole-induced dose-dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non-target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole-dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical-specific interaction and is also mediated by oxidative species.

Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats.

Cyproterone acetate (CPA), a synthetic hormonal drug, induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats compared to females. In order to evaluate the potential of the in vivo alkaline Comet assay to predict the sex-specific carcinogenicity of CPA, CPA-induced direct DNA damage (DNA strand breaks and alkali-labile sites) were evaluated in the livers of both male and female F344 rats. In addition, secondary oxidative DNA damage was measured concurrently utilizing the human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified in vivo alkaline Comet assays and the reticulocyte micronucleus (MN) frequency was analyzed in peripheral blood. Groups of 5 seven-week-old male and female F344 rats received olive oil or 10, 25, 50 or 100 mg/kg bw CPA in olive oil by gavage at 0, 24, and 45 h and were sacrificed at 48 h. CPA-induced direct DNA damage in rat liver showed the same sex-specific pattern as its hepatotumorigenicity: a five-fold-higher dose of CPA was needed to induce a statistically significant increase in direct DNA damage in livers of males compared to females. However, peripheral blood MN frequency was weak in both sexes and CPA-induced oxidative DNA damage was generally greater in male than female rat livers. Taken together, our results demonstrate concordance in the sex-specificity of CPA in the in vivo alkaline Comet assay and cancer bioassay, while the induction of oxidative DNA damage by CPA was not directly correlated with its tumorigenicity.

Early biomarkers of doxorubicin-induced heart injury in a mouse model.

Cardiac troponins, which are used as myocardial injury markers, are released in plasma only after tissue damage has occurred. Therefore, there is a need for identification of biomarkers of earlier events in cardiac injury to limit the extent of damage. To accomplish this, expression profiling of 1179 unique microRNAs (miRNAs) was performed in a chronic cardiotoxicity mouse model developed in our laboratory. Male B6C3F1 mice were injected intravenously with 3mg/kg doxorubicin (DOX; an anti-cancer drug), or saline once a week for 2, 3, 4, 6, and 8weeks, resulting in cumulative DOX doses of 6, 9, 12, 18, and 24mg/kg, respectively. Mice were euthanized a week after the last dose. Cardiac injury was evidenced in mice exposed to 18mg/kg and higher cumulative DOX dose whereas examination of hearts by light microscopy revealed cardiac lesions at 24mg/kg DOX. Also, 24 miRNAs were differentially expressed in mouse hearts, with the expression of 1, 1, 2, 8, and 21 miRNAs altered at 6, 9, 12, 18, and 24mg/kg DOX, respectively. A pro-apoptotic miR-34a was the only miRNA that was up-regulated at all cumulative DOX doses and showed a significant dose-related response. Up-regulation of miR-34a at 6mg/kg DOX may suggest apoptosis as an early molecular change in the hearts of DOX-treated mice. At 12mg/kg DOX, up-regulation of miR-34a was associated with down-regulation of hypertrophy-related miR-150; changes observed before cardiac injury. These findings may lead to the development of biomarkers of earlier events in DOX-induced cardiotoxicity that occur before the release of cardiac troponins.

Print media coverage around failed and successful tobacco tax initiatives: the South Carolina experience.

PURPOSE: Taxes are a critical tobacco control policy, yet little systematic research has determined how mass media represent tobacco taxes. This study aimed to characterize print media coverage of tobacco tax initiatives in South Carolina (SC). DESIGN: Content analysis. SETTING: The setting comprised 346 news articles from 2006 to 2010 in the four main SC newspapers. SUBJECTS: N/A . MEASURES: A coding scheme with good inter-rater reliability (α = .90-1.0) assessed article type (news vs. opinion), arguments, and the story tendency regarding whether it was in favor of the tax, against the tax, or neutral/mixed. ANALYSIS: Chi-square tests and t-tests assessed hypotheses regarding the prevalence and number of different arguments and article tendencies across different time periods (i.e., legislature in session vs. not in session; successful vs. unsuccessful initiative) and article types. RESULTS: Most articles were favorable toward the tax (59%), with favorable articles most likely to be found in opinion pieces than in news articles. Compared to unsuccessful tax initiative years (2006 to 2009), articles from the successful year (2010) were more likely to include pro-tax arguments about how the tax can raise state revenues (47% vs. 33%; p = .020) and pay for tobacco control programs (40% vs. 26%; p = .014). Unsuccessful years included a relatively higher percentage of stories about the lack of consensus regarding how the tax money should be spent (25% vs. 11%; p = .014). Within articles, the mean number of arguments favorable toward the tax and the mean number of economic arguments were marginally higher in the successful year compared to the unsuccessful years. CONCLUSION: Study results suggest that advocates build consensus and communicate more clearly on how tobacco tax revenue streams should be spent.

Toxicity evaluation of bisphenol A administered by gavage to Sprague Dawley rats from gestation day 6 through postnatal day 90.

Bisphenol A (BPA) is a high production volume industrial chemical to which there is widespread human oral exposure. Guideline studies used to set regulatory limits detected adverse effects only at doses well above human exposures and established a no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day. However, many reported animal studies link BPA to potentially adverse effects on multiple organ systems at doses below the NOAEL. The primary goals of the subchronic study reported here were to identify adverse effects induced by orally (gavage) administered BPA below the NOAEL, to characterize the dose response for such effects and to determine doses for a subsequent chronic study. Sprague Dawley rat dams were dosed daily from gestation day 6 until the start of labor, and their pups were directly dosed from day 1 after birth to termination. The primary focus was on seven equally spaced BPA doses (2.5-2700 µg/kg bw/day). Also included were a naïve control, two doses of ethinyl estradiol (EE2) to demonstrate the estrogen responsiveness of the animal model, and two high BPA doses (100,000 and 300,000 µg/kg bw/day) expected from guideline studies to produce adverse effects. Clear adverse effects of BPA, including depressed gestational and postnatal body weight gain, effects on the ovary (increased cystic follicles, depleted corpora lutea, and antral follicles), and serum hormones (increased serum estradiol and prolactin and decreased progesterone), were observed only at the two high doses of BPA. BPA-induced effects partially overlapped those induced by EE2, consistent with the known weak estrogenic activity of BPA.

Urinary biomarkers track the progression of nephropathy in hypertensive and obese rats.

AIMS: To determine whether urinary biomarkers of acute kidney injury can be used to monitor the progression of chronic kidney injury in a rat model of hypertension and obesity. MATERIALS & METHODS: A suite of novel urinary biomarkers were used to track the progression of kidney damage in SHROB and SHR-lean rats. RESULTS: Urinary albumin, NAG, clusterin, osteopontin, RPA-1 and fibrinogen levels were significantly elevated over time and were closely associated with the severity of histopathologically determined nephropathy in both SHROB and SHR-lean rats. CONCLUSION: Urinary biomarkers, such as albumin, fibrinogen, NAG, clusterin, RPA-1 and osteopontin, may serve as useful tools to track the progression of chronic kidney disease associated with hypertension and obesity.

Serum myoglobin, but not lipopolysaccharides, is predictive of AMPH-induced striatal neurotoxicity.

Determinants of amphetamine (AMPH)-induced neurotoxicity are poorly understood. The role of lipopolysaccharides (LPS) and organ injury in AMPH-induced neurotoxicity was examined in adult male Sprague-Dawley rats that were give AMPH and became hyperthermic during the exposure. Environmentally-induced hyperthermia (EIH) in the rat was compared to AMPH to determine whether AMPH-induced increases in LPS and peripheral toxicities were solely attributable to hyperthermia. Muscle, liver, and kidney function were determined biochemically at 3h or 1 day after AMPH or EIH exposure and histopathology at 1 day after treatment. Circulating levels of LPS were monitored (via limulus amoebocyte coagulation assay) during AMPH or EIH exposure. Blood LPS levels were detected in 40-50% of the AMPH and EIH rats, but the presence of LPS in the serum had no effect on organ damage or striatal dopamine depletions (neurotoxicity). In both CR and NCTR rats, serum bound urea nitrogen and creatinine levels increased at 3h after EIH or AMPH (2- to 3-fold above control) but subsided by 1 day. Alanine transaminase was increased (indicating liver dysfunction) by both AMPH and EIH at 3 h (2- to 10-fold above control) in CR rats, but the levels were not significantly different between the control and AMPH groups in NCTR animals. Mild liver necrosis was detected in 1 of 7 rats examined in the AMPH group and in 1 of 5 rats examined in the EIH group (only NCTR rats were examined). Serum myoglobin increased (indicating muscle damage) in both CR and NCTR rats at 3h and was more pronounced with AMPH (≈5-fold above control) than EIH. Our results indicate that: (1) "free" blood borne LPS often increases with EIH and AMPH but may not be necessary for striatal neurotoxicity and CNS immune responses; (2) liver or kidney dysfunction may result from muscle damage; however, it is not sufficient nor necessary to produce, but may exacerbate, neurotoxicity; (3) AMPH-induced serum myoglobin release is a potential biomarker and possibly a factor in AMPH-induced toxicity processes.

Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F1 mouse model.

Serum levels of cardiac troponins serve as biomarkers of myocardial injury. However, troponins are released into the serum only after damage to cardiac tissue has occurred. Here, we report development of a mouse model of doxorubicin (DOX)-induced chronic cardiotoxicity to aid in the identification of predictive biomarkers of early events of cardiac tissue injury. Male B6C3F(1) mice were administered intravenous DOX at 3mg/kg body weight, or an equivalent volume of saline, once a week for 4, 6, 8, 10, 12, and 14weeks, resulting in cumulative DOX doses of 12, 18, 24, 30, 36, and 42mg/kg, respectively. Mice were sacrificed a week following the last dose. A significant reduction in body weight gain was observed in mice following exposure to a weekly DOX dose for 1week and longer compared to saline-treated controls. DOX treatment also resulted in declines in red blood cell count, hemoglobin level, and hematocrit compared to saline-treated controls after the 2nd weekly dose until the 8th and 9th doses, followed by a modest recovery. All DOX-treated mice had significant elevations in cardiac troponin T concentrations in plasma compared to saline-treated controls, indicating cardiac tissue injury. Also, a dose-related increase in the severity of cardiac lesions was seen in mice exposed to 24mg/kg DOX and higher cumulative doses. Mice treated with cumulative DOX doses of 30mg/kg and higher showed a significant decline in heart rate, suggesting drug-induced cardiac dysfunction. Altogether, these findings demonstrate the development of DOX-induced chronic cardiotoxicity in B6C3F(1) mice.

Pathologic findings associated with delayed death in nonhuman primates experimentally infected with Zaire Ebola virus.

Zaire Ebola virus infection in macaques causes a fatal disease with a pathogenesis similar to that in humans. During several independent therapy studies, we noted altered tissue tropism in 6 rhesus macaques that survived longer than those with a typical disease course. The mean time to death for these 6 macaques was 21.7 days, which is significantly longer than the average mean time to death of 8.3 days for 20 untreated historical control animals. In addition to living significantly longer, these 6 animals exhibited a variety of deteriorating clinical signs with pathologic findings that were not seen in the untreated control animals, as well as the presence of viral antigen in the brain, eye, pancreas, thyroid, and lung. We suggest that treatment extended the time course of the disease and permitted the virus to infect tissues not usually affected in the typical model.

Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys.

Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.