Measurement of the oxidation state of mitochondrial cytochrome c from the neocortex of the mammalian brain.

Diffuse optical remission spectra from the mammalian neocortex at visible wavelengths contain spectral features originating from the mitochondria. A new algorithm is presented, based on analytically relating the first differential of the attenuation spectrum to the first differential of the chromophore spectra, that can separate and calculate the oxidation state of cytochrome c as well as the absolute concentration and saturation of hemoglobin. The algorithm is validated in phantoms and then tested on the neocortex of the rat during an anoxic challenge. Implementation of the algorithm will provide detailed information of mitochondrial oxygenation and mitochondrial function in physiological studies of the mammalian brain.

Cardiomegaly is a common arrhythmogenic substrate in adult sudden cardiac deaths, and is associated with obesity.

BACKGROUND: Both coronary artery disease (CAD) and cardiomyopathy may present with sudden cardiac death (SCD). It is generally accepted that CAD is the most common cause of SCD in adults, but the frequency of cardiomegaly as a primary or contributing cause is less known. METHODS: We retrospectively studied the cardiac findings of all cases of adult SCD attributed to cardiomegaly, severe atherosclerosis, or both, in the absence of specific cardiomyopathy. Association between findings and risk factors was studied. RESULTS: There were 484 sudden cardiac deaths, of which 402 met study criteria. Mean age was 49 ±â€Š13 years, with 289 men and 159 African Americans (AAs). Cardiomegaly with presumed hypertensive, multifactorial or unknown cause, was the sole arrhythmogenic substrate in 38% of men and 49% of women (p = 0.003); CAD was the sole cause of SCD in 19% of men and 26% of women, and mixed CAD + cardiomegaly the cause of death in 43% of men and 25% of women. Cardiomegaly was associated by univariate analysis with younger age (46 ±â€Š12 years versus 53 ±â€Š14 for CAD, p < 0.0001), AA race (p = 0.004), and body mass index (p < 0.0001). CONCLUSIONS: Among adults with a mean age of about 50 years, cardiomegaly is a frequent cause of sudden cardiac death, and is highly associated with obesity. Cardiomegaly is also frequent in SCD with severe CAD. The causes and classification of cardiomegaly in patients without specific cardiomyopathy, and in patients with and without hypertension or coronary disease need to be better studied.

Measurement of the mitochondrial membrane potential and pH gradient from the redox poise of the hemes of the bc1 complex.

The redox potentials of the hemes of the mitochondrial bc(1) complex are dependent on the proton-motive force due to the energy transduction. This allows the membrane potential and pH gradient components to be calculated from the oxidation state of the hemes measured with multi-wavelength cell spectroscopy. Oxidation states were measured in living RAW 264.7 cells under varying electron flux and membrane potential obtained by a combination of oligomycin and titration with a proton ionophore. A stochastic model of bc(1) turnover was used to confirm that the membrane potential and redox potential of the ubiquinone pool could be measured from the redox poise of the b-hemes under physiological conditions assuming the redox couples are in equilibrium. The pH gradient was then calculated from the difference in redox potentials of cytochrome c and ubiquinone pool using the stochastic model to evaluate the ΔG of the bc(1) complex. The technique allows absolute quantification of the membrane potential, pH gradient, and proton-motive force without the need for genetic manipulation or exogenous compounds.

Spectral components of the α-band of cytochrome oxidase.

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.

Death due to bioterrorism-related inhalational anthrax: report of 2 patients.

On October 9, 2001, a letter containing anthrax spores was mailed from New Jersey to Washington, DC. The letter was processed at a major postal facility in Washington, DC, and opened in the Senate's Hart Office Building on October 15. Between October 19 and October 26, there were 5 cases of inhalational anthrax among postal workers who were employed at that major facility or who handled bulk mail originating from that facility. The cases of 2 postal workers who died of inhalational anthrax are reported here. Both patients had nonspecific prodromal illnesses. One patient developed predominantly gastrointestinal symptoms, including nausea, vomiting, and abdominal pain. The other patient had a "flulike" illness associated with myalgias and malaise. Both patients ultimately developed dyspnea, retrosternal chest pressure, and respiratory failure requiring mechanical ventilation. Leukocytosis and hemoconcentration were noted in both cases prior to death. Both patients had evidence of mediastinitis and extensive pulmonary infiltrates late in their course of illness. The durations of illness were 7 days and 5 days from onset of symptoms to death; both patients died within 24 hours of hospitalization. Without a clinician's high index of suspicion, the diagnosis of inhalational anthrax is difficult during nonspecific prodromal illness. Clinicians have an urgent need for prompt communication of vital epidemiologic information that could focus their diagnostic evaluation. Rapid diagnostic assays to distinguish more common infectious processes from agents of bioterrorism also could improve management strategies.

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model.

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.

Sterol regulatory element-binding protein-1 participates in the regulation of fatty acid synthase expression in colorectal neoplasia.

Endogenous fatty acid synthesis has been observed in certain rapidly proliferating normal and neoplastic tissues. Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of lipogenic genes including fatty acid synthase (FAS), the major biosynthetic enzyme for fatty acid synthesis. We have previously shown that SREBP-1, FAS, and Ki-67, a proliferation marker, colocalized in the crypts of the fetal gastrointestinal tract epithelium. This study sought to determine whether SREBP-1 participates in the regulation of proliferation-associated fatty acid synthesis in colorectal neoplasia. An immunohistochemical analysis of SREBP-1, FAS, and Ki-67 expression in 25 primary human colorectal carcinoma specimens showed colocalization in 22 of these. To elucidate a functional linkage between SREBP-1 activation and proliferation-associated FA synthesis, SREBP-1 and FAS content were assayed during the adaptive response of cultured HCT116 colon carcinoma cells to pharmacological inhibition of FA synthesis. Cerulenin and TOFA each inhibited the endogenous synthesis of fatty acids in a dose-dependent manner and each induced increases in both precursor and mature forms of SREBP-1. Subsequently, both the transcriptional activity of the FAS promoter in a luciferase reporter gene construct and the FAS expression increased. These results demonstrate that tumor cells recognize and respond to a deficiency in endogenous fatty acid synthesis by upregulating both SREBP-1 and FAS expression and support the model that SREBP-1 participates in the transcriptional regulation of lipogenic genes in colorectal neoplasia.

Cholesterol embolization in renal allografts.

Renal cholesterol embolization (RCE) in native kidneys has a dismal outcome and frequently leads to irreversible renal failure. RCE may rarely occur in renal allografts as well, particularly if the recipient or the donor has prominent atherosclerosis. The natural history of RCE in renal transplants is unknown. We have reviewed the surgical pathology files of The Johns Hopkins Hospital in the 14-year period between 1984 and early 1999 and found 7 RCE cases among 1500 renal transplant biopsies (0.47%). One of the seven cases had three biopsies showing cholesterol emboli, the first of which was a postreperfusion (immediate posttransplant) biopsy. The probable source of the cholesterol emboli was the recipient in six cases and the donor in one case. Five donors were cadaveric and two were living donors. Six biopsies were taken within the first 4 months posttransplant (four were postreperfusion biopsies). One recent patient had the inciting event of arteriography and stent placement 2 years posttransplant and is currently doing well. One kidney failed due to posttransplant lymphoproliferative disorder (PTLD), another kidney failed with complicating opportunistic infections, and the other five were functioning 2 to 6 years posttransplant. A literature review revealed additional 14 RCE cases in renal transplants. Combining our cases with those in the literature (21 cases), reveals that the origin of the RCE was probably the recipient in 11 cases (seven cadaveric, two living-related, and two unknown), and the donor in 10 cases (eight cadaveric and two unknown). Graft failure occurred in two of the 11 cases, where RCE was of probable recipient origin. Seven of the 10 kidneys, where the RCE was probably of donor origin, failed due to allograft dysfunction; one of them also developed superimposed rejection and cytomegalovirus infection. We conclude that if RCE is originating in the recipient, graft survival is usually good. In contrast, if RCE is of donor origin, graft dysfunction and subsequent graft loss are common. The reason for this difference may be the more extensive RCE developing in an atherosclerotic cadaveric donor during organ procurement or severe trauma leading to death.

Needle biopsy of recurrent adenocarcinoma of the prostate after radical prostatectomy.

The objective of this study was to evaluate needle biopsy of recurrent prostate cancer after radical prostatectomy. We evaluated 37 cases of recurrent prostate cancer after radical prostatectomy that were diagnosed by needle biopsy between March 1984 and July 1998. Fifteen were from consultations in which contributors were uncertain of the diagnosis, and 22 were from men who had come to The Johns Hopkins Hospital for treatment. The median interval from radical prostatectomy to biopsy showing recurrent tumor was 40 months. There was no correlation between the interval to recurrence and either pathologic features of the biopsy and radical prostatectomy or various clinical features. The mean extent of adenocarcinoma in the biopsies was 3.2 mm (range, 0.1 to 18 mm; median, 2 mm). The length of recurrent cancer on biopsy correlated with an abnormal rectal examination (P = .001). The mean Gleason score for the recurrent tumors was 6.5, which correlated with the grade of the radical prostatectomy cancer (P = .005). The cancers often lacked overt histologic features of malignancy. Benign prostatic acini were seen in five cases (14%), usually separate from the cancer. In 5 (33%) of the consultation cases, we would not have been able to diagnose cancer if not for the fact that atypical prostate glands should not be present after radical prostatectomy. In well-sampled radical prostatectomies, margins were almost always positive, as was extraprostatic extension. In cases with less sampling, there was a higher incidence of organ-confined disease and margin-negative disease implying suboptimal processing of the radical prostatectomy. After radical prostatectomy, recurrent cancer on needle biopsies may be focal and difficult to diagnose and must be assessed differently than in patients who have not had surgery.

Lethal combination of tramadol and multiple drugs affecting serotonin.

The death of a 36-year-old alcoholic man who died after developing seizure activity while being treated with tramadol, as well as with venlafaxine, trazodone, and quetiapine, all of which interact with the neurotransmitter serotonin, is reported. The decedent, who had a history of chronic back pain, alcoholism, depression, mild hypertensive cardiovascular disease, and gastritis, had just been discharged from the hospital after 4 days of alcohol detoxification treatment. During the admission, no withdrawal seizures were noted. The morning after discharge, a witness observed the decedent exhibiting seizure activity and then collapsing. An autopsy was performed approximately 6 hours after death, and the anatomic findings were consistent with seizure activity and collapse, which included biting injuries of the tongue and soft-tissue injuries of the face. Toxicologic analysis identified tramadol, venlafaxine, promethazine, and acetaminophen in the urine; tramadol (0.70 mg/L) and venlafaxine (0.30 mg/L) in the heart blood, and 0.10 mg of tramadol in 40 ml of submitted stomach contents. No metabolites, such as acetate, acetone, lactate, and pyruvate, were found in the specimens that would be characteristically found in a person with alcohol withdrawal syndrome. The threshold for seizures is lowered by tramadol. In addition, the risk for seizure is enhanced by the concomitant use of tramadol with selective serotonin reuptake inhibitors or neuroleptics, and its use in patients with a recognized risk for seizures, i.e., alcohol withdrawal. The cause of death in this individual was seizure activity complicating therapy for back pain, depression, and alcohol withdrawal syndrome. The data in Adverse Event Reporting System of the Food and Drug Administration from November 1, 1997 to September 8, 1999 was reviewed along with a MEDLINE search from 1966 to the present. This case appears to be the first reported death caused by seizure activity in a patient taking tramadol in combination with drugs that affect serotonin.

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells.

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well.

Effect of antioxidants on androgen-induced AP-1 and NF-kappaB DNA-binding activity in prostate carcinoma cells.

BACKGROUND: Previous studies have suggested that male hormones (androgens) and certain forms of oxygen (reactive oxygen species) are linked to the development of prostate cancer. We hypothesized that androgens contribute to prostate carcinogenesis by increasing oxidative stress. We further hypothesized that antioxidants reduce prostate cancer risk by modulating androgen effects on cellular processes. METHODS: To test these hypotheses, we looked for 1) a change in the level of reactive oxygen species in the presence of androgens, 2) androgen-induced binding activity of transcriptional activators AP-1 and NF-kappaB, whose activities are known to be altered during cell proliferation, and 3) the effect of antioxidants on androgen-induced transcription factor binding. RESULTS: Physiologic concentrations (1 nM) of 5alpha-dihydrotestosterone or 1-10 nM R1881, a synthetic androgen, produced sustained elevation of AP-1 and NF-kappaB DNA-binding activity in LNCaP cells, an androgen-responsive human prostate carcinoma cell line. Androgen-independent DU145 cells (another human prostate carcinoma cell line) were unaffected by R1881 treatment. AP-1-binding activity increased 5 hours after 1 nM R1881 treatment; NF-kappaB DNA-binding activity increased after 36 hours. Both activities remained elevated for at least 120 hours. Nuclear AP-1 and NF-kappaB protein levels were not elevated. Antioxidant vitamins C plus E blocked both androgen-induced DNA-binding activity and production of reactive oxygen species. CONCLUSION: Physiologic concentrations of androgens induce production of reactive oxygen species and cause prolonged AP-1 and NF-kappaB DNA-binding activities, which are diminished by vitamins C and E.

Androgen-induced oxidative stress in human LNCaP prostate cancer cells is associated with multiple mitochondrial modifications.

We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.

Alteration in gamma-glutamyl transpeptidase activity and messenger RNA of human prostate carcinoma cells by androgen.

Concentrations of the synthetic androgen R1881 that correspond to physiologically relevant concentrations of 5alpha-dihydrotestosterone are capable of altering the activity of gamma-glutamyl transpeptidase (GGT) in human prostate carcinoma cells. GGT activity of the androgen-responsive prostate cancer cell line LNCaP increases >50% above that of the control after a 72-h exposure to 1 nM R1881. This elevation in GGT activity occurs as early as 48 h after treatment and is maintained for at least 96 h. Loss of glutathione (GSH) from media and accumulation of intracellular GSH of cells pretreated with 1 nM R1881 occur at a higher rate than in control cells, suggesting that a greater rate of GSH salvage is associated with the increased GGT activity. Immunohistochemical staining detects an increase in GGT-positive staining in cells treated with 1 nM R1881 for 72 h. Steady-state mRNA levels for GGT are elevated above those of the control 24-72 h after treatment. R1881 has no effect on the GGT activity of the androgen-independent prostate cell line DU145. Growth of LNCaP but not DU145 cells is inhibited by 1 nM R1881 compared to that of the control. Inhibitors of GGT activity, acivicin and serine-borate, are capable of dampening or blocking the effect of R1881 on growth. Growth of LNCaP cells treated with 1 nM R1881 plus 100 mM glycylglycine, a stimulator of GGT activity, is inhibited to a greater extent than the growth of LNCaP cells treated with R1881 alone. These data demonstrate that androgens can elevate GGT activity and increase GGT mRNA and protein levels in human prostate carcinoma cells. In addition, compounds able to alter GGT activity are capable of altering androgen-related growth effects.

Prooxidant-antioxidant shift induced by androgen treatment of human prostate carcinoma cells.

BACKGROUND: Prostate cancer is a disease associated with aging. Also commonly associated with increasing age is a shift in the prooxidant-antioxidant balance of many tissues toward a more oxidative state, i.e., increased oxidative stress. We hypothesize that androgen exposure, which has long been associated with the development of prostate cancer, may be a means by which the prooxidant-antioxidant balance of prostate cells is altered. PURPOSE: Using established prostate carcinoma cell lines, we studied the effect of androgens on various parameters of oxidative state (e.g., generation of hydrogen peroxide and hydroxyl radicals, lipid peroxidation, and oxygen consumption) and antioxidant defense mechanisms (e.g., the glutathione system and catalase). METHODS: The androgen-responsive LNCaP and the androgen-independent DU145 prostate carcinoma cell lines were exposed to 5 alpha-dihydrotestosterone (DHT) and to the synthetic androgen R1881. The cellular proliferation responses were measured by use of a fluorometric assay to quantitate the amount of DNA. The generation of reactive oxygen species was measured by use of 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals. Lipid peroxidation was quantitated by use of a chromogen specific for malonaldehyde and 4-hydroxy-2(E)-nonenal. General mitochondrial activity was determined by assaying 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. A Clark-type electrode was used to assess oxygen consumption per cell. Intracellular glutathione concentrations and the activities of catalase and gamma-glutamyl transpeptidase were measured spectrophotometrically. All P values resulted from two-sided tests. RESULTS: DHT at less than 1 to 100 nM (a concentration range encompassing the physiologic levels of DHT considering all ages) and R1881 at 0.1-1 nM concentrations were effective in inducing in LNCaP cells comparable proliferative responses and changes in oxidative stress. In contrast, neither DHT nor R1881 had any effect on the oxidative stress in DU145 cells. The mitochondrial activity in LNCaP cells, as measured by MTT reduction, was significantly elevated above the levels of the untreated controls by DHT (0.1-1000 nM) and R1881 (0.05-1 nM) (P < .001 in both). Oxygen consumption and catalase activity were increased in LNCaP cells in the presence of 1 nM R1881 by 60% and 40%, respectively, over the values in the untreated control cells (P < .03 and P < .01, respectively). The same concentration of R1881 resulted in a decrease in intracellular glutathione concentrations and an increase in gamma-glutamyl transpeptidase activity in LNCaP cells. Treatment with the oxidizing agents H2O2 and menadione produced an increase in gamma-glutamyl transpeptidase activity in LNCaP cells, whereas treatment with the antioxidant compound ascorbic acid (100 mM) reduced the oxidative stress produced in LNCaP cells by 1 nM R1881 and completely blocked the gamma-glutamyl transpeptidase activity. CONCLUSIONS: Physiologic levels of androgens are capable of increasing oxidative stress in androgen-responsive LNCaP prostate carcinoma cells. The evidence suggests that this result is due in part to increased mitochondrial activity. Androgens also alter intracellular glutathione levels and the activity of certain detoxification enzymes, such as gamma-glutamyl transpeptidase, that are important for maintenance of the cellular prooxidant-antioxidant balance.

Adult-onset energy restriction of rhesus monkeys attenuates oxidative stress-induced cytokine expression by peripheral blood mononuclear cells.

We previously reported that energy restriction (ER) of mice attenuated age-associated increases in serum levels of interleukin-6 (IL-6). Here, we studied peripheral blood mononuclear cells (PBMC) from male rhesus monkeys to investigate the following: 1) the production of IL-6 and other cytokines become dysregulated with aging; 2) ER influences cytokine production and mRNA expression; and, 3) oxidative stress, as induced in vitro by xanthine and xanthine oxidase (X/XOD), influences cytokine mRNA and protein levels. Two types of comparisons were made as follows: 1) between normally fed young (6-9 y) and old monkeys (22-33 y); and 2) between middle-aged monkeys (15-21 y) fed either a normal energy intake or subjected to ER (for 5.5 y at 30% less than base-line intake). IL-6 protein levels and X/XOD-induced IL-6 mRNA levels in PBMC from old monkeys were significantly greater than those in PBMC from young animals. In contrast, interleukin-1beta (IL-1beta) and interleukin-8 mRNA levels were not strongly influenced by advancing age. X/XOD, which increased levels of protein carbonyls (indicative of oxidative damage) in PBMC, induced the expression of all three cytokines. ER reduced IL-6 protein and mRNA levels induced by X/XOD and the unstimulated mRNA levels of IL-1beta. These results indicate that, in a nonhuman primate model, oxidative stress may contribute to age-associated increases in the levels of certain cytokines and that adult-onset ER partially ameliorates this alteration.

Alteration of glyceraldehyde-3-phosphate dehydrogenase activity and messenger RNA content by androgen in human prostate carcinoma cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) activity and mRNA content is altered in the androgen-responsive human prostate carcinoma cell line LNCaP after exposure to the synthetic androgen R1881. Elevation in GAPDH activity is noted as early as 24 h after treatment with 1 nM R1881 and lasts at least 96 h. R1881 has no effect on the activity of GAPDH in androgen-independent DU145 cells. LNCaP GAPDH mRNA content is lowered by treatment with 1 nM R1881; the magnitude of reduction appears to depend on the length of exposure. The results present at least one means by which androgen-responsive tissues may develop alterations in GAPDH mRNA or activity, as is found in certain tumor tissues.

Characteristics of the glutathione/glutathione-S-transferase detoxification system in melphalan resistant human prostate cancer cells.

Glutathione (GSH) and glutathione-S-transferases (GST) have been implicated in resistance of tumor cells to certain alkylating agents, including melphalan. Glutathione levels and GST activities were determined in melphalan-resistant sublines of the human prostate carcinoma cell lines DU 145, PC-3 and LNCaP produced by serial treatment with melphalan at progressively increasing concentrations. The resistant sublines M4.5DU145, M5DU145, M6DU145, M6PC-3 and M6LNCaP were 27-, 7-, 3-, 6- and 2-fold more resistant to melphalan than the parental lines. The melphalan-resistant DU 145 and PC-3 lines showed cross-resistance to cisplatin and tetraplatin, but retained sensitivity to vinblastine, colchicine and etoposide. Interestingly, both sublines were also resistant to methotrexate and adriamycin. The melphalan-resistant LNCaP line showed slight resistance to cisplatin and adriamycin, but remained sensitive to tetraplatin and methotrexate. This line also retained sensitivity to vinblastine while developing resistance to colchicine. Intracellular GSH levels were increased 2.8 fold for M5DU145, 1.7 fold for M6PC-3 and 2.1 fold for M6LNCaP compared to the parental lines, whereas GST activity using chlorodinitro-benzene as a substrate was comparable for all lines. When cumene hydroperoxide was used as a substrate, an increase in GST activity was noted only in the M6PC-3 line as compared with the parent line. Western blot analysis showed no change in GST isozyme profile between parent and resistant DU 145 lines; however a mu class isoenzyme was detected in the resistant, but not in the parent PC-3 line, using a Yb1 antibody. M5DU145 cells maintained in the absence of melphalan for seven months maintained their resistance to melphalan. Depletion of GSH, with buthionine sulfoximine, to control levels reversed melphalan resistance to control levels.

Detection of cocaine and its metabolites in human amniotic fluid.

The dramatic rise in maternal drug abuse and the incidence of positive drug findings during neonatal testing has increased the need for prenatal toxicological testing for drugs of abuse. Human amniotic fluid samples collected after 13-39 weeks of pregnancy were screened for cocaine metabolite (benzoylecgonine) by fluorescence polarization immunoassay (FPIA). All positive samples, as well as any accompanying maternal serum, were confirmed by gas chromatography/mass spectrometry (GC/MS) for cocaine and its metabolites. Five samples out of 450 were positive for cocaine, benzoylecgonine, and ecgonine methyl ester by GC/MS. In addition, one sample was also positive for cocaethylene. Two maternal serum samples were positive for benzoylecgonine and ecgonine methyl ester. The presence of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene in the amniotic fluid suggests that the fetus is exposed to cocaine and its metabolites through maternal circulation. The impact of this exposure on the health of the newborn is unknown.