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Significance The biology of HIV DNA, from its synthesis to its integration into the host genome, remains poorly understood. Here we show that in the nucleus, histones are rapidly loaded on newly synthesized unintegrated HIV DNA. Interestingly, the chromatin architecture around the HIV long terminal repeat (LTR) is different in unintegrated and integrated HIV DNA. Specifically, a nucleosome present only on the DNase hypersensitive site of unintegrated HIV DNA contributes to the transcriptional silencing of unintegrated HIV DNA by preventing RNAPII recruitment., The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. We found that histones are loaded on HIV-1 DNA after its nuclear import and before its integration in the host genome. Nucleosome positioning analysis along the unintegrated and integrated viral genomes revealed major differences in nucleosome density and position. Indeed, in addition to the well-known nucleosomes Nuc0, Nuc1, and Nuc2 loaded on integrated HIV-1 DNA, we also found NucDHS, a nucleosome that covers the DNase hypersensitive site, in unintegrated viral DNA. In addition, unintegrated viral DNA-associated Nuc0 and Nuc2 were positioned slightly more to the 5′ end relative to their position in integrated DNA. The presence of NucDHS in the proximal region of the long terminal repeat (LTR) promoter was associated with the absence of RNAPII and of the active histone marks H3K4me3 and H3ac at the LTR. Conversely, analysis of integrated HIV-1 DNA showed a loss of NucDHS, loading of RNAPII, and enrichment in active histone marks within the LTR. We propose that unintegrated HIV-1 DNA adopts a repressive chromatin structure that competes with the transcription machinery, leading to its silencing.

This study enables a better understanding of how nucleation-growth of zeolites affects glass dissolution kinetics in hyperalkaline solutions characteristic of cement waters. A 20-oxide borosilicate glass, an inactive surrogate of a typical intermediate level waste glass, was altered in static mode at 50 °C in a hyperalkaline solution rich in Na+, K+ and Ca2+ and at an initial pH50°C of 12.6. Experiments were performed at four glass-surface-area-to-solution-volume (S/V) ratios to investigate various reaction progresses. Two types of glass alteration kinetics were obtained: (i) at low S/V, a sharp alteration resumption occurred after a rate drop regime, (ii) at high S/V, a high dissolution rate was maintained throughout the test duration with a slight progressive slow-down. In all the experiments, zeolites precipitated but the time taken to form stable zeolite nuclei varied dramatically depending on the S/V. Resulting changes in pH affected zeolite composition, morphology, solubility and growth rate. A change in a critical parameter such as S/V affected all the processes controlling glass dissolution.

Perfluorooctanoic acid (PFOA), a global environmental pollutant detected in both wildlife and human populations, has several pathophysiological effects in experimental animals, including hepatotoxicity, immunotoxicity, and developmental toxicity. However, details concerning the tissue distribution of PFOA, in particular at levels relevant to humans, are lacking, which limits our understanding of how humans, and other mammals, may be affected by this compound. Therefore, we characterized the tissue distribution of 14C-PFOA in mice in the same manner as we earlier examined its analogues perfluorooctanesulfonate (PFOS) and perfluorobutanesulfonate (PFBS) in order to allow direct comparisons. Following dietary exposure of adult male C57/BL6 mice for 1, 3 or 5 days to a low dose (0.06 mg/kg/day) or a higher experimental dose (22 mg/kg/day) of 14C-PFOA, both scintillation counting and whole-body autoradiography revealed the presence of PFOA in most of the 19 different tissues examined, demonstrating its ability to leave the bloodstream and enter tissues. There were no differences in the pattern of tissue distribution with the low and high dose and the tissue-to-blood ratios were similar. At both doses, PFOA levels were highest in the liver, followed by blood, lungs and kidneys. The body compartments estimated to contain the largest amounts of PFOA were the liver, blood, skin and muscle. In comparison with our identical studies on PFOS and PFBS, PFOA reached considerably higher tissue levels than PFBS, but lower than PFOS. Furthermore, the distribution of PFOA differed notably from that of PFOS, with lower tissue-to-blood ratios in the liver, lungs, kidneys and skin.

High-dose exposure of mice to perfluorooctanoate (PFOA) induces both hepatotoxicity and immunotoxicity. Here, we characterized the effects of 10-day dietary treatment with PFOA (0.002-0.02%, w/w) on the liver and complement system of male C57BL/6 mice. At all four doses, this compound caused hepatomegaly and reduced the serum level of triglycerides (an indicator for activation of the peroxisome proliferator-activated receptor-alpha (PPARα)). At the highest dose (0.02%, w/w), this hepatomegaly was associated with the hepatic injury, as reflected in increased activity of alanine aminotranferase (ALAT) in the serum, severe hepatocyte hypertrophy and hepatocellular necrosis. PFOA-induced hepatic injury was associated with in vivo activation of the complement system as indicated by (i) significant attenuation of the serum activities of both the classical and alternative pathways; (ii) a marked reduction in the serum level of the complement factor C3; and (iii) deposition of the complement factor C3 fragment (C3a) in the hepatic parenchyma. PFOA did not activate the alternative pathway of complement in vitro. At doses lower than 0.02%, PFOA induced hepatocyte hypertrophy without causing liver injury or activating complement. These results reveal substantial involvement of activation of complement in the pathogenesis of PFOA-induced hepatotoxicity.

Numerous studies have been conducted on glass and cement durability in contact with water, but very little work to date has focused directly on interactions between the two materials. These interactions are mostly controlled by silicon–calcium reactivity. However, the physical and chemical processes involved remain insufficiently understood to predict the evolution of coupled glass–cement systems used in several industrial applications. Results are reported from borosilicate glass alteration in calcium-rich solutions. Our data show that four distinct behaviors can be expected according to the relative importance of three key parameters: the pH, the reaction progress (short- or long-term alteration) and the calcium concentration. Glass alteration is thus controlled by specific mechanisms depending on the solution chemistry: calcium complexation at the glass surface, precipitation of calcium silicate hydrates (C–S–H) or calcium incorporation in the altered layer. These findings highlight the impact of silicon–calcium interactions on glass durability and open the way for a better understanding of glass–cement mixing in civil engineering applications as well as in nuclear waste storage.

Exposure of mice to perfluorooctanoate (PFOA) evokes pronounced hepatomegaly along with significant alterations in both the histological structure and immune status of the liver. The present study was designed to evaluate the effects of this perfluorochemical on immune-mediated liver damage. In this connection, the influence of both sub-acute (10 days), moderate-dose (0.002% w/w = 3 ± 0.7 mg/kg body weight/day) and short-term (28 days), low-dose (0.00005% w/w = 70 ± 2 μg/kg body weight/day) dietary pretreatment with PFOA on the development of concanavalin A (Con A)-induced liver damage in mice was examined. With sub-acute, moderate, but not short-term, low-dose exposure, PFOA aggravated the acute liver damage caused by Con A, i.e., elevated serum levels of transaminases and led to more pronounced damage of hepatic tissue. This aggravation was associated with significantly enhanced hepatic level of interleukin-6 (IL-6), but unaltered hepatic levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-4 (IL-4). Moreover, hepatic DNA fragmentation was not changed by sub-acute exposure to the moderate-dose. Our findings imply that exposure to PFOA may sensitize hepatic parenchymal cells to other toxicants that activate the hepatic immune system and thereby aggravate liver injury during acute inflammation.

It is well established that exposure of mice to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) exerts adverse effects on the thymus and spleen. Here, we characterize the effects of a 10-day dietary treatment with these compounds (0.001–0.02%, w/w) on the bone marrow (BM) of mice. At a dose of 0.02%, both compounds reduced food consumption and caused atrophy of the thymus and spleen. At this same dose, histopathological and flow cytometric analysis revealed that (i) the total numbers of BM as well as the numbers of myeloid, pro/pre B, immature B and early mature B cells were all reduced significantly; and (ii) these adverse effects were reversed either partially or completely 10 days after withdrawal of these compounds. At the lower dose of 0.002%, only PFOA reduced the B-lymphoid cell population. Finally, mice fed an amount of diet equivalent to that consumed by the animals exposed to 0.02% PFOA also exhibited atrophy of the thymus and spleen, and a reduction in the number of B-lymphoid population, without affecting myeloid cells. Thus, in mice, immunotoxic doses of PFOA or PFOS induce adverse effects on the myeloid and B-lymphoid cells in the BM, in part as a consequence of reduced food consumption.

The widespread environmental pollutant perfluorooctane sulfonate (PFOS), detected in most animal species including the general human population, exerts several effects on experimental animals, e.g. ...

It is now well established that the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARα) is expressed in different types of immune cells and plays a pivotal role in the regulation of age-related production of inflammatory cytokines. However, the role(s) of this receptor in the regulation of immune cell homoeostasis in ageing non-lymphoid and lymphoid organs has not yet been resolved. We examine this issue here by evaluating the hepatic and splenic immune status and immunoglobulin (Ig) production in male PPARα-null mice and their wild-type littermates at one and 2 years of age. In comparison with the age-matched control animals, PPARα-null mice exhibited age-related elevations in the numbers of total, as well as of phenotypically distinct subpopulations of intrahepatic immune cells (IHIC) and splenocytes. Moreover, at 2 years of age, these alterations in hepatic immune cells were accompanied by significant increases in hepatic levels of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interferon-gamma (IFN-γ), in combination with the development of hepatic inflammatory loci containing mixtures of leucocytes. Alterations in splenocytes of old PPARα-null mice were also accompanied by increases in cellularity of both white and red pulps of the spleen. Furthermore, these same animals exhibited pronounced increases in the numbers of splenic plasma cells and enhanced production of Ig of different isotypes, including IgG1, IgG2a and IgE. Thus, our findings indicate that upon ageing, PPARα plays a crucial role in regulating the total numbers, compositions and functions of immune cells in both lymphoid and non-lymphoid immune organs of mice.

Exposure of rodents in utero to perfluorooctane sulfonate (PFOS) impairs perinatal development and survival. Following intravenous or gavage exposure of C57Bl/6 mouse dams on gestational day (GD) 16 to (35)S-PFOS (12.5mg/kg), we determined the distribution in dams, fetuses (GD18 and GD20) and pups (postnatal day 1, PND1) employing whole-body autoradiography and liquid scintillation counting. In dams, levels were highest in liver and lungs. After placental transfer, (35)S-PFOS was present on GD18 at 2-3 times higher levels in lungs, liver and kidneys than in maternal blood. In PND1 pups, levels in lungs were significantly higher than in GD18 fetuses. A heterogeneous distribution of (35)S-PFOS was observed in brains of fetuses and pups, with levels higher than in maternal brain. This first demonstration of substantial localization of PFOS to both perinatal and adult lungs is consistent with evidence describing the lung as a target for the toxicity of PFOS at these ages.

The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.

Introduction We designed this phase II trial to evaluate the efficacy and safety of weekly paclitaxel in combination with monthly carboplatin as first-line treatment in elderly patients with advanced non-small cell lung cancer (NSCLC). Methods Main eligibility criteria were histologically or cytologically proven stage IIIB or IV NSCLC, age ≥70 years, Eastern Cooperative Oncology Group performance status 0-2, and measurable disease. The 4-week–based chemotherapy regimen consisted of carboplatin infusion (area under the concentration-time curve 6 mg/ml -1 /min) on day 1 and paclitaxel 90 mg/m 2 as a 1-hour infusion on days 1, 8, and 15. Tumor response was evaluated using Response Evaluation Criteria in Solid Tumors criteria, and symptoms were evaluated using the Lung Cancer Symptoms Scale. Analyses were performed on an intention-to-treat basis. Results From February 2002 to August 2003, 51 patients (median age, 74 years) participated in the study. One complete and 21 partial responses were reported by the independent review committee, leading to an intention-to-treat objective response rate of 43% (95% confidence interval, 30–57%). The median progression-free and overall survivals were 7.5 (95% confidence interval, 6.2–9.4) and 13.6 (95% confidence interval, 7.5–17) months, respectively. Longitudinal evaluation of the Lung Cancer Symptoms Scale demonstrated lack of quality of life modification during the treatment period. Neurotoxicity was mild to moderate, with 6% of patients suffering from a grade 3 or 4 neuropathy. Myelosuppression was the main toxicity; 39% of patients experienced grade 3 or 4 neutropenia, 18% experienced grade 3 anemia, and 8% experienced grade 3 or 4 thrombocytopenia. There was no treatment-related death. Conclusions The combination of weekly paclitaxel 90 mg/m 2 administered on days 1, 8, and 15 plus monthly carboplatin area under the curve 6 on day 1 of a 4-week cycle was feasible and active as a first-line treatment for elderly patients with NSCLC with a good safety profile. These results deserve further analysis to compare the standard care for these patients (monotherapies) with this doublet.

Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.

We have previously demonstrated that severe adipose tissue atrophy occurs upon dietary treatment of mice with potent peroxisome proliferators (PPs). This atrophy occurs subsequent to peroxisome proliferation in the liver and may represent a novel addition to the pleiotropic effects exerted by PPs. In the present study we have characterized the recovery of mice from such atrophy following cessation of exposure. Following termination of treatment with perfluorooctanoic acid (PFOA) for 7 days, the adipose tissue atrophy was rapidly reversed, beginning on 2-5 days of recovery and being complete within 10 days. In contrast, hepatic peroxisome proliferation recovered much more slowly, indicating that these processes are not strictly coordinated. Analysis of lipoprotein lipase and hormone-sensitive lipase activities in adipose tissue revealed that the decrease and increase in these activities, respectively, caused by PFOA were both reversed within 10 days of recovery. Overall, these data provide further support for our previous conclusion that the adipose tissue atrophy induced by PFOA is caused, at least in part, by changes in the activities of lipoprotein lipase and hormone-sensitive lipase. The serum level of cholesterol, which increased after termination of PFOA treatment, returned to normal with a time-course similar to the recovery of adipose tissue weight, although hepatic peroxisome proliferation was still present. The possible relationship between the reduction in serum cholesterol and/or in its availability to peripheral tissues and the associated atrophy of adipose tissues caused by PPs is discussed.

Dimethyl sulfoxide (DMSO) is widely and routinely used as a vehicle in various investigations, especially within the pharmaceutical industry. It has been used for the evaluation of the effects of hydrophobic xenobiotics on cells, as well as for the cryopreservation of biological material. Isothermal microcalorimetry is a powerful tool for monitoring heat production, which is a function of general cellular metabolic activity. Employing this microcalorimetric technique, a low concentration of DMSO routinely used for the addition of hydrophobic substances to, e.g., cell cultures, was shown to decrease heat production (per unit DNA) by the rat hepatoma cell lines FAO, Morris 7800C1 and H4IIE by 32–38%. However, such low concentrations of DMSO did not influence the cell cycle or the degree of apoptosis in these cell populations. Caution is thus advisable when utilizing DMSO as a vehicle in cell culture experiments.

In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111–118; L. Staffas, L. Mankowitz, M. Soderstrom, A. Blanck, I. Porsch-Hallstrom, C. Sundberg, B. Mannervik, B. Olin, J. Rydstrom, and J.W. DePierre, Biochem J, 1992, 286, 65–72) isoenzymes of GST, primarily of the μ class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45–50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:169–173, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10034

To evaluate whether preoperative chemotherapy (PCT) could improve survival in resectable stage I (except T1N0), II, and IIIA non-small-cell lung cancer (NSCLC).A randomized trial compared PCT to primary surgery (PRS). PCT consisted of two cycles of mitomycin (6 mg/m(2), day 1), ifosfamide (1.5 g/m(2), days 1 to 3) and cisplatin (30 mg/m(2), days 1 to 3), and two additional postoperative cycles for responding patients. In both arms, patients with pT3 or pN2 disease received thoracic radiotherapy.Three hundred fifty-five eligible patients were randomized. Overall response to PCT was 64%. There were two preoperative toxic deaths. Postoperative mortality was 6.7% in the PCT arm and 4.5% in the PRS arm (P =.38). Median survival was 37 months (95% confidence interval [CI], 26.7 to 48.3) for PCT and 26.0 months (95% CI, 19.8 to 33.6) for PRS (P =.15). Survival differences between both arms increased from 3.8% (95% CI, 1.3% to 25.1%) at 1 year to 8.6% (95% CI, 2.64% to 24.4%) at 4 years. A quantitative interaction between N status and treatment was observed, with benefit confined to N0 to N1 disease (relative risk [RR], 0.68; 95% CI, 0.49 to 0.96; P =.027). After a nonsignificant excess of deaths during treatment, the effect of PCT was significantly favorable on survival (RR, 0.74; 95% CI, 0.56 to 0.99; P =.044). Disease-free survival time was significantly longer in the PCT arm (P =.033).Although impressive differences in median, 3-year, and 4-year survival were observed, they were not statistically significant, except for stage I and II disease.

The regulation of the aldo-keto reductase AKR7A1 was examined in the livers of male and female rats during development by using Western blots, and its contribution to carbonyl metabolism was assessed by using enzyme assays. Hepatic levels of AKR7A1 are low in fetal rats and rise to a peak at around 6 weeks of age in animals of both sexes. Higher levels of the enzyme are found in adult male rat liver than in adult female rat liver. The reductase, therefore, appears to be subject to sex-specific regulation. The effect of growth hormone in mediating this difference in expression was examined by using hypophysectomized animals whose serum growth hormone levels had been feminized by continuous administration. Results demonstrate that such treatment leads to a reduction in AKR7A1 expression. AKR7A1 was found to be constitutively expressed in rat tissues such as liver, kidney, small intestine, and testis, but it was not detected in nasal mucosa, skeletal muscle, heart, adrenal gland, brain, or spleen. However, AKR7A1 was inducible by the synthetic antioxidant ethoxyquin in liver, kidney, and small intestine, but not in the other tissues examined. These results show that levels of this important detoxication enzyme vary considerably according to age and sex and that dietary antioxidants can also influence its level in several tissues.

A dose escalation study of paclitaxel in combination with vinorelbine was conducted in 21 patients with previously untreated stage IIIb or IV non-small cell lung cancer (NSCLC). All three patients treated with the initial dose of paclitaxel 135 mg/m2 administered as a 1-h intravenous infusion and vinorelbine 25 mg/m2 experienced dose-limiting toxicity (febrile neutropenia). After modification of the dosing schedule, the MTD of paclitaxel was found to be 115 mg/m2 when combined with vinorelbine 20 mg/m2 on day 1, followed by vinorelbine 20 mg/m2 on day 5. Partial responses were achieved in 24% of patients, with a median duration of response of 126 days (range from 84 to 484 days) and a 1-year survival rate of 42%. In conclusion, haematologic toxicity (febrile neutropenia/neutropenia) severely restricts the dosing schedule of combined paclitaxel and vinorelbine, and possibly limits anti-tumour efficacy.

The present study concerns conjugation of 1-naphthol in primary cultures of rat ovarian cells. Two phase II enzymes catalyzing conjugation, i.e. phenol sulfotransferase (P-SULT) and phenol UDP-glucuronosyltransferase (P-UGT), were measured using 1-naphthol as substrate. The rates of conjugation by the different cell types of the rat ovary were the same at low concentrations and short incubation times. However, after 20 h of incubation the rate of conjugation in cells isolated from ovaries enriched in corpora lutea (CL) exceeded the rate in cells isolated from ovaries enriched in preovulatory follicles. In addition, when the granulosa cells were removed from the preovulatory follicles, the rate of conjugation was 1.7-fold higher, i.e. in the theca/stroma cells. When the cells were incubated with 1-[14C]naphthol and conjugates were subsequently separated by thin-layer chromatography, naphthyl glucuronide was the only conjugate observed. Pentachlorophenol (PCP), a commonly used inhibitor of P-SULT, inhibited 1-naphthol conjugation 50% in cell cultures, as well as in microsomal preparations. alpha-Naphthoflavone (ANF) and ellipticine (ELP), both cytochrome P450 (CYP) inhibitors, affected the conjugation of 1-naphthol in different ways; ANF did not affect P-UGT activity in microsomal preparations, but inhibited 1-naphthol conjugation in cell cultures by as much as 90%. On the other hand, ELP inhibited the conjugation of 1-naphthol up to 99% in the cell cultures, but only 75% in microsomal fractions. Testosterone (TST) and estradiol inhibited this activity approximately equal 50% in both of these experimental systems. Clomiphene citrate (CLF), a drug used to induce ovulation and demonstrating both estrogenic and antiestrogenic effects, did not influence the conjugation of 1-naphthol significantly in the cell cultures. The present findings demonstrate that P-UGT is by far the major enzyme conjugating 1-naphthol in the rat ovary and that commonly used inhibitors of P-SULT and CYPs also inhibit P-UGT activity, either directly or via other mechanisms.

The levels and subcellular distribution of enzymes involved in defenses against reactive oxygen superoxide dismutase (SOD; E.C.1.15.1.1), glutathione peroxidase (GPX; E.C.1.11.1.9), catalase (CAT; E.C.1.11.1.6), and DT-diaphorase (DT; E.C.1.6.99.2) and of the conjugating enzymes glutathione transferase (GST; E.C.2.5.1.18) and p-sulphotransferase (p-ST; E.C.2.8.2.1) in the corpus luteum of ovaries from pregnant and non-pregnant pigs were investigated. In addition, non-protein thiols and glutathione reductase (GRD; E.C.1.6.4.2) were examined in the same manner. The total cytosolic activities of CAT, DT, GRD, and p-ST were significantly increased, whereas total GST activity was decreased in the pregnant corpus luteum compared to the corresponding activities in non-pregnant corpus luteum. In the case of the mitochondrial fraction from pregnant corpus luteum, GPX and GRD displayed significant increases in specific activity. Upon subfractionation of the mitochondrial fraction (i.e. mitoplast preparation), SOD activity was distributed equally between the mitoplasts and the supernatant. CAT and GPX activities were mainly recovered in the supernatant, while the major GRD activity pelleted with the mitoplasts. Microsomes from pregnant corpus luteum demonstrated increased specific GPX activity and decreased SOD activity compared to the non-pregnant corpus luteum. No differences in the non-protein thiol levels in the cytosolic, mitochondrial, or microsomal fractions from the corpus luteum were observed between non-pregnant and pregnant sows.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.

In the present study we have employed a microcalorimetric procedure to measure the heat generated by a porcine renal tubule cell line (LLC-PK1) and its Na+, K+-ATPase. Microplates with an area of 2.2 cm2 were found to be optimal in terms of producing sufficient heat and a steady-state power curve. We compared the rate of heat production by cells in suspension and on monolayers and found a much lower value in suspension, that is, 1.42+/-0.2 versus 2.54+/-0.19 microW/microg DNA. Ouabain, the specific Na+, K+-ATPase inhibitor, caused a reduction in this heat output. The maximal inhibition in cell suspensions was 40% and remained unchanged with as much as 100 microM ouabain, the highest concentration tested. With cells cultured on microplates, ouabain in the concentration interval 0.1-3 microM caused a 25% inhibition of heat output. With 25-100 microM ouabain, a 50% inhibition was observed and at higher concentrations, no further inhibition occurred. Furthermore, upon removal of ouabain, full recovery of the Na+, K+-ATPase was observed, a process that could easily be monitored by using cell monolayers cultured on microplates.

Apoptosis is a form of programmed cell death that is involved in cell turnover. In the present study we show that the tricyclic antidepressants (TCAS) imipramine, clomipramine and citalopram induce apoptosis in human peripheral lymphocytes. Lymphocytes were incubated with these three drugs for up to 48 h. Apoptosis was characterized by typical nucleosomal DNA fragmentation on agarose gel, as well as quantitated using 4′-6-diamidino-2-phenylindole (DAPI) staining and 3′-OH end-labeling of fragmented DNA at the single cell level. Apoptosis induced by TCAs was shown to be dose-dependent and could be detected after a 24 h incubation. The optimal concentrations of the three TCAs found to induce apoptosis were 50 μ M imipramine, 20 μ M clomipramine and 180 μ M citalopram. Furthermore, immunofluorescence and three-color flow cytometry were used to identify the phenotype of apoptotic cells. TCA-induced apoptosis was shown to involve exclusively T-lymphocytes. Cytotoxic T-lymphocytes were more prone to undergo apoptosis than were T-helper cells. In conclusion, the present investigation clearly demonstrates that TCAs exert cell biological effects upon human T-lymphocytes. Further studies are required to determine the possible clinical relevance of these findings.

A novel and rapid procedure for the isolation of catalase from mouse liver, after prior treatment with the peroxisome proliferator perfluorooctanoic acid was developed using immobilized metal ion affinity chromatography involving chelation with zinc ions. The purification developed is simple, rapid (requiring 3 hours from cytosol or peroxisomal matrix to homogeneous proteins), reproducible, and yields virtually complete overall recovery of catalase activity. This procedure makes catalase from a variety of tissues and physiological and environmental conditions more readily available for study.

We have previously found that tricyclic antidepressants (TCAs) induce apoptosis in quiescent human lymphocytes. The aim of the present study was to evaluate if TCAs induce apoptosis in proliferating human lymphocytes and in established blastoid lymphocytes also. The development of conA-induced lymphoblast populations was followed by measuring the CD25 membrane expression. Three TCA compounds were run with the following concentrations: imipramine (10, 20, 30, 40, 60 microM), clomipramine (1, 10, 20, 30, 40 microM) and citalopram (40, 60, 80, 100, 180 microM). They all induced a dose-dependent apoptosis both in continuously transformed, as well as in established lymphoblasts. Preincubation of the TCA up to 48 h did not significantly increase induction of apoptosis. The three drugs tested were found to be potent inducers of apoptosis in proliferating lymphocytes. Furthermore, we found that the apoptotic populations in proliferating and in established blastoid lymphocytes were of fairly the same magnitude than in the corresponding population in TCA-incubated resting lymphocytes. In conclusion, we demonstrate that TCAs induce apoptosis in proliferating lymphocytes, as they do in quiescent lymphocytes. Furthermore, the extent of apoptosis was even more pronounced in TCA-incubated lymphoblasts compared to TCA-treated resting lymphocytes.

This study was designed to investigate the expression of different isoenzymes of glutathione transferase (GST) in the rat ovary and to follow possible changes in the pattern of expression during maturation and the different stages of the oestrus cycle. The GST subunits present in the rat ovary (as analyzed by HPLC of affinity-purified GSTs) were A3, A4, M1, M2, M3 and P1. The most abundant subunit in the ovary was A3, but the mu enzymes demonstrated the largest increase (3.5-fold) when the mature ovary was compared to the immature organ. An overall decrease in GST isoenzyme content during dioestrus was observed, but there were no other recurrent changes during the oestrus cycle. Treatment of immature rats with pregnant mare's serum gonadotropin clearly demonstrated that the mu and alpha isoenzymes are up-regulated by gonadotropin stimulation, i.e. a 4-6 fold increase was seen. This treatment also elevated the P1 subunit 3-fold. At present, it is only possible to speculate concerning the mechanism(s) underlying these variations in ovarian GST expression in connection with hormonal changes. The functional significance of these variations is not yet known.

Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.

Thirty-one patients with previously untreated advanced non-small cell lung cancer were included in a Phase I study to determine the optimal dose of Carboplatin (CBDCA) which preserves the best Navelbine (NVB) dose-intensity. NVB was administered at a 30-mg/m 2 fixed-dose on days 1–8/q 3 weeks, whereas CBDCA doses were planned to be escalated from 275 to 400 mg/m 2 on day 1/q 3 weeks for six successive groups of patients. The toxicity limiting dose of CBDCA in the combination was 350 mg/m 2 on day 1/q 3 weeks because of repetitive Grade IV neutropenia, and the optimal dose of CBDCA was 325 mg/m 2 on day 1/q 3 weeks, offering a 86.4% NVB and a 92.6% CBDCA relative dose-intensity for the first 9 weeks. Responses were observed at each step. This study demonstrates the feasibility and the efficacy of the NVB-CBDCA combination. It suggests that dose-intensity calculation can be helpful to determine the recommended dose for Phase II studies of new drug combinations.

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe3+ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H2O2 in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and alpha-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.

Perfluorobutanesulfonyl fluoride (PBSF) has been introduced as a replacement for its eight-carbon homolog perfluorooctanesulfonyl fluoride (POSF) in the manufacturing of fluorochemicals. Fluorochemicals derived from PBSF may give rise to perfluorobutanesulfonic acid (PFBS) as a terminal degradation product. Although basic mammalian toxicokinetic data exist for PFBS, information on its tissue distribution has only been reported in one study focused on rat liver. Therefore, here we characterized the tissue distribution of PFBS in mice in the same manner as we earlier examined its eight-carbon homolog perfluorooctanesulfonate (PFOS) to allow direct comparisons. Following dietary exposure of adult male C57/BL6 mice for 1, 3 or 5d to 16 mg (35)S-PFBS kg(-1) d(-1), both scintillation counting and whole-body autoradiography (WBA) revealed the presence of PFBS in all of the 20 different tissues examined, demonstrating its ability to leave the bloodstream and enter tissues. After 5d of treatment the highest levels were detected in liver, gastrointestinal tract, blood, kidney, cartilage, whole bone, lungs and thyroid gland. WBA revealed relatively high levels of PFBS in male genital organs as well, with the exception of the testis. The tissue levels increased from 1 to 3 d of exposure but appeared thereafter to level-off in most cases. The estimated major body compartments were whole bone, liver, blood, skin and muscle. This exposure to PFBS resulted in 5-40-fold lower tissue levels than did similar exposure to PFOS, as well as in a different pattern of tissue distribution, including lower levels in liver and lungs relative to blood.

Exposure of rodents to perfluorooctane sulfonate (PFOS) induces pronounced hepatomegaly associated with significant alterations in hepatic histophysiology and immune status. The present investigation was designed to evaluate the effects of this perfluorochemical on immune-mediated liver damage. Accordingly, the influence of both sub-acute (10 days), moderate-dose (0.004%, w/w = 6 ± 1.3 mg/kg body weight/day) or short-term (28 days), low-dose (0.0001%, w/w = 144 ± 4 μg/kg body weight/day) dietary pretreatment with PFOS on the development of concanavalin A (Con A)-induced liver damage in mice was examined. With either regimen of exposure, PFOS exacerbated the acute liver damage caused by Con A, i.e., elevated serum levels of transaminases and led to more pronounced damage of hepatic tissue. This exacerbation was associated with either reduced (moderate dose) or unaltered (low dose) hepatic levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). Moreover, hepatic DNA fragmentation was enhanced, particularly following short-term exposure to a low-dose. Our findings suggest that exposure to PFOS may sensitize hepatic parenchymal cells to other insults that activate the hepatic immune system and thereby exacerbate liver damage during acute inflammation.

In the French geological repository concept, intermediate-level vitrified wastes could be disposed of in a cement medium. The glass dissolution mechanisms and kinetics, expected to depend strongly on the chemical composition and pH of the leaching water, were studied in various cement pore water compositions corresponding to different stages of cement aging. In this study, we focused on the effects induced by cement pore water at equilibrium with respect to Portlandite (pH25°C = 12.4). A decrease in the maximum glass dissolution rate due to the effect of calcium was clearly observed compared to the reference medium, i.e., at the same pH in KOH solution. At higher reaction progress, calcium in solution was almost totally consumed after a few days, probably due to the formation of Calcium Silicate Hydrate (C-S-H) phases with silicon leached from the glass. Two assumptions can be proposed to explain the effect of calcium on the initial regime: either the calcium from solution reacts with silicon released to form a C-S-H passivating layer at the glass/solution interface, or calcium compensates two nonbridging oxygen (Si–O−) in the altered layer, which could decrease the hydrolysis of silicon bonds.

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.

Here, we describe for the first time the synthesis of [(35)S] PFOS and [(35)S] PFBS with sulfur-35 enriched sulfur dioxide as the radiolabelled reagent, resulting in 2.5 and 2.3 mCi of product, respectively. Basic information concerning the physicochemical properties of perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate (PFBS) and perfluorooctanoic acid (PFOA) are still limited. Hence, we utilized these radiolabelled perfluoroalkanesulfonates (PFSAs), as well as carbon-14 labelled perfluorooctanoic acid ([(14)C] PFOA) to determine some basic characteristics of physiological and experimental significance. The solubility of PFOS in buffered aqueous solutions at pH 7.4 was found to be severely reduced in the presence of potassium and sodium ions, which, however, did not reduce the solubility of PFOA or PFBS. PFOS was found to adhere to a small extent to polypropylene and polystyrene, whereas no such adhesion of PFOA or PFBS was detected. The extents of adhesion of PFOS and PFOA to glass were found to be 20% and 10%, respectively. For the first time, the partition coefficients for PFOS, PFBS and PFOA between n-octanol and water were determined experimentally, to be -0.7, -0.3, and 1.4, respectively, reflecting the difference in the amphiphilic natures of these molecules.

The capacity of ovarian granulosa cells from rat and pig to release reactive metabolites produced from polycyclic aromatic hydrocarbons with the ability to cause mutations in neighbouring cells has been studied. For this purpose we have used cocultivation with V79 Chinese hamster cells as a detection system. The cells were treated with two different polycyclic aromatic hydrocarbons (PAHs), 7,12-dimethylbenz(a)anthracene (DMBA) or (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol). The resulting mutation frequency in the V79 cells after cocultivation, as a function of granulosa cell number, DMBA, or BP-7,8-diol concentration and time, was determined. The progesterone concentration in the medium after cocultivation was also analyzed as a measure of the differentiation of the granulosa cells. These studies demonstrate an increasing frequency of mutations in the V79 cells with an increasing number of granulosa cells. The maximal number of mutations were achieved with a DMBA or BP-7,8-diol concentration of 5 or 2 microM, respectively. The optimal cocultivation time was 24 h. These results clearly show that the granulosa cells can bioactivate PAHs to reactive metabolites with the capacity to migrate into surrounding cells and cause mutations in these cells. Compounds metabolized to mutagenic products by granulosa cells might thus cause mutations in the neighbouring germ cells, with possible consequences for the offspring.

It is well established that exposure of mice to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) induces hepatomegaly and, concurrently, immunotoxicity. However, the effects of these perfluorochemicals on the histology and immune status of the liver have not been yet investigated and we have examined these issues here. Dietary treatment of male C57BL/6 mice with 0.002% (w/w) PFOA or 0.005% (w/w) PFOS for 10 days resulted in significant reductions in serum levels of cholesterol and triglycerides, a moderate increase in the serum activity of alkaline phosphatase (ALP) and hepatomegaly, without affecting other immune organs. This hepatomegaly was associated with marked hypertrophy of the centrilobular hepatocytes, with elevated numbers of cytoplasmic acidophilic granules and occasional mitosis. Furthermore, dietary exposure to PFOA or PFOS altered the hepatic immune status: whereas exposure to PFOA enhanced the numbers of total, as well as of phenotypically distinct subpopulations of intrahepatic immune cells (IHIC), and in particular the presumptive erythrocyte progenitor cells, treatment with PFOS enhanced only the numbers of hepatic cells that appear immunophenotypically to be erythrocyte progenitors, without affecting other types of IHIC. In addition, exposure to these compounds attenuated hepatic levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-4 (IL-4). Furthermore, the exposed animals exhibited a significant increase in hepatic levels of erythropoietin, a hormone required for erythropoiesis. Thus, in mice, PFOA- and PFOS-induced hepatomegaly is associated with significant alterations in hepatic histophysiology and immune status, as well as induction of hepatic erythropoiesis.

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.

Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2-10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2-10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subfraction. The most dramatic changes observed were a 5-9-fold increase in mitochondrial protein, a 3-6-fold increase in the microsomal content of cytochrome P450, a 3-10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.

Male C57Bl/6 mice were treated for 5 days with 0.05% perfluorooctanoic acid (PFOA) in their diet. This treatment resulted in a potent induction of peroxisomal fatty acid beta-oxidation in the liver. In order to investigate recovery from treatment with PFOA, mice were given normal laboratory chow for up to 20 days after termination of PFOA administration. It was established that the activities of peroxisomal lauoryl-CoA oxidase and palmitoyl-CoA oxidation were still elevated 2-3 weeks after termination of treatment. The catalase activity recovered in the cytosolic fraction was also still significantly elevated after 20 days with normal laboratory chow. Furthermore, the protein content of the mitochondrial fraction was increased by PFOA and had not returned to control level at the end of the recovery period. Perfluorooctanoic acid also caused a persistent effect in omega hydroxylation of lauric acid (cytochrome P-452). The activities of cytosolic DT-diaphorase and glutathione transferase were also enhanced by PFOA. However, these two enzymes recovered relatively rapidly from the treatment (2-20 days). This study reveals two different patterns of recovery from PFOA treatment, one involving parameters that recovered completely, or almost completely, from PFOA treatment after 20 days and another involving parameters that were still elevated at the end of the recovery period.

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.

Short-term exposure of mice to high doses of perfluorooctanesulfonate (PFOS), an ubiquitous and highly persistent environmental contaminant, induces various metabolic changes and toxic effects, including immunotoxicity. However, extrapolation of these findings to the long-term, low-dose exposures to which humans are subject is highly problematic. In this connection, recent studies have concluded that sub-chronic (28-day) exposure of mice by oral gavage to doses of PFOS that result in serum levels comparable to those found in general human populations suppress adaptive immunity. Because of the potential impact of these findings on environmental research and monitoring, we have examined here whether sub-chronic dietary exposure (a major route of human exposure) to a similarly low-dose of PFOS also suppress adaptive immune responses. Dietary treatment of male B6C3F1 mice for 28 days with a dose of PFOS that resulted in a serum concentration of 11mug/ml (ppm) significantly reduced body weight gain and increased liver mass. However, this treatment did not alter the cellular compositions of the thymus and spleen; the number of splenic cells secreting IgM antibodies against sheep red blood cell (SRBC); serum levels of IgM and IgG antibodies specifically towards SRBC; or circulating levels of IgM antibodies against the T-cell-independent antigen trinitrophenyl conjugated to lipopolysaccharide (TNP-LPS). These findings indicate that such sub-chronic dietary exposure of mice to PFOS resulting in serum levels approximately 8-85-fold greater than those observed in occupationally exposed individuals does not exert adverse effects on adaptive immunity.

Having found previously that high-dose, short-term dietary exposure of mice to perfluorooctanesulfonate (PFOS) or perfluorooctanoate (PFOA) suppresses adaptive immunity, in the present study we characterize the effects of these fluorochemicals on the innate immune system. Male C57BL/6 mice receiving 0.02% (w/w) PFOS or PFOA in their diet for 10 days exhibited a significant reduction in the numbers of total white blood cells (WBC), involving lymphopenia in both cases, but neutropenia only in response to treatment with PFOA. Moreover, both compounds also markedly reduced the number of macrophages (CD11b(+) cells) in the bone marrow, but not in the spleen or peritoneal cavity. The ex vivo production of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) by peritoneal macrophages isolated from animals treated with PFOA or PFOS was increased modestly. Moreover, both fluorochemicals markedly enhanced the ex vivo production of these same cytokines by peritoneal and bone marrow macrophages stimulated either in vitro or in vivo with lipopolysaccharide (LPS); whereas there was no such effect on splenic macrophages. The serum levels of these inflammatory cytokines observed in response to in vivo stimulation with LPS were elevated substantially by prior exposure to PFOA, but not by PFOS. None of these parameters of innate immunity were altered in animals receiving a dietary dose of these compounds that was 20-fold lower (0.001%, w/w). These findings reveal that in addition to suppressing adaptive immunity, high-dose, short-term exposure of mice to either PFOS or PFOA augments inflammatory responses to LPS, a potent activator of innate immunity.

Myelin bodies were isolated from the renal cortex of gentamicin-treated rats (100 mg/kg body weight, twice daily for 3 days, i.p.) employing an initial pelleting by differential centrifugation and subsequent flotation on a discontinuous sucrose gradient. These structures were found to contain almost twice as much protein as phospholipid and SDS-polyacrylamide gel electrophoresis revealed the presence of many different polypeptides. All the major phospholipids are present, although myelin bodies contain a considerably higher proportion of phosphatidylinositol, somewhat more phosphatidylcholine and considerably lower percentages of phosphatidylserine and sphingomyelin than do normal renal phospholipids. The fatty acids of myelin body phospholipids are highly saturated (67.3-87.9%) and a striking feature is the occurrence of relatively large amounts of 22:1, presumably erucic acid, especially in sphingomyelin. Myelin bodies contain small amounts of unesterified cholesterol, unesterified dolichol and coenzymes Q9 and Q10.

After hypophysectomy, the level of glutathione transferase subunit 4 increases in the adrenal, as well as in the liver, as do those of several other forms of glutathione transferase. This increase in subunit 4 can subsequently be down-regulated by administration of adrenocorticotropin. The present investigation demonstrates that also in primary cultures of female rat adrenal cells an increase in the level of glutathione transferase subunit 4 (as shown by immunoblotting) occurs in the absence of adrenocorticotropin. When adrenocorticotropin or dibutyryladenosine 3',5'-phosphate was administered to these cells, a down-regulation of this enzyme level was observed, in agreement with the in vivo situation. This down-regulation was not affected by aminoglutethimide, an inhibitor of the cholesterol-side-chain-cleavage enzyme (cytochrome P-450scc) which is the rate-limiting step in the biosynthesis of steroids. Hence adrenal steroid production is not involved in the down-regulation of glutathione transferase subunit 4 by adrenocorticotropin.