Carcinogenic potency of perfluorooctane sulfonate (PFOS) on Syrian hamster embryo (SHE) cells.

Perfluorooctane sulfonate (PFOS) is the degradation product of many fluoroderivatives and a widespread environmental contaminant. Its persistence, its long half-life in humans and its toxicity explain high concerns on human health side effects in future. PFOS is suspected to be a non-genotoxic carcinogen. In the present work, we assessed carcinogenic potential of PFOS by studying morphological transformation in Syrian hamster embryo (SHE) cells; cell transformation of SHE cells is an in vitro assay recommended by the Organization for Economic Cooperation and Development to detect carcinogens, genotoxic or not. Genotoxicity of PFOS and expression of PPARs genes in SHE cells were also measured. PFOS was shown to induce cell transformation (P < 0.05) at non-cytotoxic concentrations (0.2 and 2 µg/mL) (P ≤ 0.01). No genotoxic effect was recorded in the range of PFOS concentrations tested (2 × 10(-4) to 50 µg/mL) using the single-cell gel electrophoresis (comet) assay after 5 and 24 h of exposure. The expression of PPARs genes was measured by qPCR within the first 24 h and after 7 days of PFOS treatment. Results indicated an increased expression of ppar-ß/δ isoform as early as 24 h. After 7 days, the increase of ppar-ß/δ mRNA was significant at the concentrations inducing cell transformation (0.2 and 2 µg/mL), while overexpression of ppar-γ and ppar-α did not closely relate to effective concentrations. The results indicate that PFOS behave as a non-genotoxic carcinogen and impacted PPARs genes. Its cell transforming potential paralleled an increased expression of ppar-ß/δ.

2,4-Dichlorophenoxyacetic acid: effects on Syrian hamster embryo (SHE) cell transformation, c-Myc expression, DNA damage and apoptosis.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a selective, systemic auxin-type herbicide extensively used throughout the world. The present research was aimed at studying effects of low and non-cytotoxic concentrations of 2,4-D on SHE cells in relation with carcinogenicity. Effects were studied on Syrian hamster morphological cell transformation, c-Myc expression - both at the gene and protein level - DNA damage and apoptosis. 2,4-D significantly induced cell transformation at 11.5 microM and 23 microM (i.e. 2.5 microg/mL and 5 microg/mL). An increase in the expression of the transcription factor c-Myc, measured by use of RT-PCR with respect to mRNA level and by Western blotting for protein level was registered at these concentrations, as well as genotoxic effects evaluated with the single-cell gel electrophoresis (Comet) assay. Consequences for apoptosis of 2,4-D treatment were also investigated. The fluorochrome acridine orange was used to study DNA fragmentation as a marker of apoptosis. No effect on apoptosis was found at 2,4-D concentrations that induced cell transformation. This was confirmed by the unchanged expression of Bcl-2 and Bax, two regulator genes of the mitochondrial pathway of apoptosis. Our results demonstrate the transforming and genotoxic effects of low concentrations of 2,4-D in mammalian cells. This information contributes to a better understanding of the mechanism of 2,4-D toxicity in mammalian cells and demonstrates that 2,4-D should be considered as potentially hazardous to humans.

Di-(2-ethylhexyl)phthalate (DEHP) increases Bcl-2/Bax ratio and modifies c-myc expression in Syrian hamster embryo (SHE) cells.

The objective of this work was to study the anti-apoptotic properties of the non-genotoxic rodent carcinogen, di(2-ethylhexyl)phthalate (DEHP) in Syrian hamster embryo (SHE) cells. We demonstrated that a 24 h pre-treatment of SHE cells with 50 microM DEHP inhibited apoptosis triggered by growth factors deprivation. The RNA expression levels of the regulator genes involved in the apoptotic pathway, bcl-2, bax and of c-myc were measured using Western blotting and RT-PCR. We showed that a 24 h treatment of SHE cells with 50 microM DEHP increased (P < 0.05) the bcl-2 expression, while c-myc expression was decreased. No effect on bax expression was observed in the range of 10-50 microM. The defective regulation of apoptosis caused by DEHP treatment could contribute to its carcinogenicity.

Comparison of early and late feline immunodeficiency virus encephalopathies.

DESIGN: The study of the early and late stages of encephalopathy following infection by the feline immunodeficiency virus (FIV) was carried out with laboratory and naturally infected cats. INTERVENTIONS: Animals infected experimentally were injected with three different isolates of the virus, administered either intracerebrally or intravenously, and sacrificed at 7 days, 1 and 6 months (intracerebral injection), and 2, 6 and 12 months (intravenous injection) post-inoculation, respectively. CONCLUSIONS: General features of encephalopathy were found to be identical, regardless of the method of inoculation or the viral strain used. Moderate gliosis and glial nodules, sometimes associated with perivascular infiltrates and white matter pallor, were observed at 1 month (intracerebral injection) and 2 months (intravenous injection), and remained unchanged until 12 months post-inoculation. The fact that these initial stages are identical for intravenously and intracerebrally inoculated cats suggests that the virus enters the brain very quickly in intravenously infected animals. Encephalopathy in cats naturally infected with FIV only consisted of gliosis, glial nodules, white matter pallor, meningeal perivascular calcification and meningitis. These lesions were more frequent and more severe in the group coinfected with feline leukaemia virus and feline infectious peritonitis virus. Although multinucleated cells were rare, the strong similarities between HIV and simian immunodeficiency virus encephalopathies at comparable stages support the view that FIV infection may represent an interesting model for a physiopathological approach of HIV infection of the central nervous system.

The presence of cryoprecipitable immunoglobulins in normal human sera may reflect specific molecular interactions.

The nature of the minute amounts of cryoprecipitable proteins present in all normal sera has been investigated in particular relation to the physicochemical and functional properties of the precipitated immunoglobulins. Cryoprecipitated material from 48 normal donor sera were first analyzed for their protein, IgG and IgM content. SDS-PAGE analysis revealed many bands in a majority of samples but in about one third a very limited number of bands was observed. IgM, IgG and albumin were identified and a lower molecular weight component (16 KD) was always present. Restricted clonality was demonstrated by IEF in 25-30% of the samples. In 50% of cryoprecipitates IgG was present in higher molecular weight fractions than 7S on sucrose density gradient at neutral pH. Ultracentrifugation at acid pH lead to a dissociation of these IgG fractions in most of the samples. A positive reaction for IgM rheumatoid factor was seen in 63% of the samples and there was a specific enrichment of RF activity in cryoprecipitates as compared to the corresponding serum. These data suggest that the cryoprecipitation of immunoglobulins in normal serum reflects specific interactions between immunoglobulin molecules rather than merely a non-specific precipitation of cold-insoluble molecules.

ZnCl2 induces Syrian hamster embryo (SHE) cell transformation.

In order to test the hypothesis of a relationship between apoptosis and neoplastic transformation, we studied the transforming potency of zinc, known for its antiapoptotic effects. In this study, zinc chloride (100 microM) was shown to induce morphological transformation (MT) in Syrian hamster embryo (SHE) cells. It was also tested in combination with benzo(a)pyrene (BaP), a positive control for carcinogenicity, or fomesafen, a carcinogenic pesticide with hepatic peroxisomal proliferation properties. A co-exposure of the two carcinogens with 100 microM zinc increased cell transformation in SHE cells. These results were in agreement with the theory of a relationship between the inhibition of apoptosis and induction of cell transformation. The cloning efficiency (CE) of SHE cells seeded at clonal density was raised by zinc, fomesafen and furthermore by the mixture of the two chemicals, which could be explained by the antiapoptotic action of zinc and fomesafen on SHE cells. No change in myc and bax expressions was observed in zinc-treated SHE cells.

Perfluorooctanoic acid (PFOA) acts as a tumor promoter on Syrian hamster embryo (SHE) cells.

Perfluorooctane sulfonate (PFOS) (C(8)F(17)SO(3)) and perfluorooctanoic acid (PFOA) (C(8)HF(15)O(2)) are synthetic chemicals widely used in industrial applications for their hydrophobic and oleophobic properties. They are persistent, bioaccumulative, and toxic to mammalian species. Their widespread distribution on earth and contamination of human serum raised concerns about long-term side effects. They are suspected to be carcinogenic through a nongenotoxic mode of action, a mechanism supported by recent findings that PFOS induced cell transformation but no genotoxicity in Syrian hamster embryo (SHE) cells. In the present study, we evaluated carcinogenic potential of PFOA using the cell transformation assay on SHE cells. The chemical was applied alone or in combination with a nontransformant concentration of benzo[a]pyrene (BaP, 0.4 µM) in order to detect PFOA ability to act as tumor initiator or tumor promoter. The results showed that PFOA tested alone in the range 3.7 × 10(-5) to 300 µM did not induce SHE cell transformation frequency in a 7-day treatment. On the other side, the combination BaP/PFOA induced cell transformation at all PFOA concentrations tested, which revealed synergistic effects. No genotoxicity of PFOA on SHE cells was detected using the comet assay after 5 and 24 h of exposure. No significant increase in DNA breakage was found in BaP-initiated cells exposed to PFOA in a 7-day treatment. The whole results showed that PFOA acts as a tumor promoter and a nongenotoxic carcinogen. Cell transformation in initiated cells was observed at concentrations equivalent to the ones found in human serum of nonoccupationally and occupationally exposed populations. An involvement of PFOA in increased incidence of cancer recorded in occupationally exposed population cannot be ruled out.

Morphological cell transformation of Syrian hamster embryo (SHE) cells by the cyanotoxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanotoxin which has been implicated in human intoxication and animal mortality. Genotoxic activity of this hepatotoxin is known but its carcinogenic activity remains to be elucidated. In this work, CYN was assessed for its cell-transforming activity using the Syrian hamster embryo (SHE) cell transformation assay. This in vitro assay is used to evaluate the carcinogenic potential of chemical, physical and biological agents in SHE cells, which are primary, normal, diploid, genetically stable and capable of metabolic activation. We demonstrated that CYN induced a significant increase in morphological cell transformation in SHE cells following a 7-day continuous treatment in the range of non-cytotoxic concentrations 1 x 10(-7)-1 x 10(-2) ng/mL.

Changes in expression of bcl-2 and bax in Syrian hamster embryo (SHE) cells exposed to ZnCl2.

Zinc is involved in many physiological processes and plays a critical role in functional and structural cells. Zinc at concentrations ranging from 100 to 150 micromol L(-1) has been shown to induce morphological transformation of Syrian hamster embryo (SHE) cells. At these concentrations, zinc inhibited apoptosis in SHE cells. The objective of this study was to elucidate the mechanisms of action of zinc on the apoptotic pathway. Effects of 100 and 150 micromol L(-1) ZnCl(2) on the expression of two members of the Bcl-2 family of proteins and on the transcription factor c-Myc in SHE cells was investigated using RT-PCR. No effect on the proto-oncogene c-myc was observed. Up-regulation of bcl-2 expression was found and bax expression was reduced. These changes have been corroborated by immunoblotting. Effects of Zn(2+) on bcl-2/bax ratio were confirmed in apoptotic camptothecin-treated SHE cells. Cloned and sequenced cDNAs obtained from RT-PCR amplifications allowed us to check the RT-PCR products encoded the expected proteins. This study demonstrated that zinc acts in the early phases of the apoptotic process by modification of the bcl-2/bax ratio in normal and apoptotic SHE cells.

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera from lepromatous patients and infected armadillos.

Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component.

Different time course patterns of local expression of delayed-type hypersensitivity to sheep red blood cells in mice.

The delayed-type hypersensitivity (DTH) reaction, a peripheral expression of cell-mediated immunity is still a crucial in vivo immunological test. Nevertheless, the biological significance of its time course remains unclear. Thus, an exhaustive study of DTH was undertaken in mice immunized with increasing doses of sheep red blood cells (SRBC) inoculated intravenously (iv) or subcutaneously. The results showed that overall DTH reactions peaked at 18 hr except in mice iv immunized with the lowest doses (10(5) and 10(6)) and elicited at Day 4. The protracted DTH reaction was shown to be associated with an histological picture of tuberculin-type reaction. A part of the 18-hr DTH reaction is mediated by serum in mice inoculated with large doses of SRBC; nevertheless, numeration by limiting dilution analysis of circulating DTH cells showed that the frequency of these cells correlates with the 18-hr DTH level. The protracted DTH shown at 42 and 48 hr, 4 days after immunization with 10(5) and 10(6) SRBC, could not be transferred in naive recipients with immune spleen cells; it was independent of the antigen life span and did not result from immunization modulation at the bone marrow level on recruitable cells.

Early SIV encephalopathy.

SIV encephalopathy was studied in rhesus macaques early after intracerebral (IC) or intravenous (IV) inoculation. Although SIV was detected in the brain of all IC-inoculated animals, the CNS showed moderate neuropathological changes. IV-inoculated animals presented a spectrum of brain changes ranging from perivascular infiltrates to multinucleated giant cells. CNS infection was detected as early as seven days post-IV-inoculation, mostly in a perivascular localization. Using combined immunohistochemistry and in situ hybridization, infected cells were shown to express macrophage markers.

Identification of components of IC purified from human sera. I. Immune complexes purified from sera of patients with SLE.

Immune complexes (IC) were purified by affinity chromatography on conglutinin columns from human sera (five SLE, one AML and one leishmaniasis) and compared with IC formed in vitro in the presence of normal serum (NHS). First, analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated a common qualitative pattern, but with marked quantitative differences, in IC obtained from five patients' sera (four SLE, one leishmaniasis) and for in vitro formed IC. In two other patients (one SLE, one AML), the pattern of IC components was very different, with a major band in the 26 kD region. Secondly, after electrophoretic transfer of the SDS-PAGE bands to nitrocellulose membranes, the nature of IC components was studied by defining the reactivity of the bands with antisera against human serum antigens. Several serum proteins were identified in the purified IC:IgG, IgA, IgM, C1q, C1r, C1s, C3bi and Bb. A few bands did not correspond with any normal serum protein. One of them, at 26 kD was shown to react with anti-C reactive protein (CRP) antiserum. From all the constituents observed in the SDS-PAGE analysis of purified IC, only two bands in one SLE patient might be corresponding to unidentified antigens.

Early viral replication in the brain of SIV-infected rhesus monkeys.

To investigate the mechanism of simian immunodeficiency virus (SIV) entry into the central nervous system (CNS) and the initial events leading to neuropathogenesis, SIV replication was studied by in situ hybridization in the CNS of 5 Rhesus macaques at 7 days, 1, 2, and 3 months after SIV intravenous inoculation. CNS infection was found to be a frequent and early event, as SIV was detected in the CNS of all the animals studied and as early as 7 days postinoculation. At the earliest stage, the infection localized mainly to perivascular cells. Using combined immunohistochemistry and in situ hybridization, infected cells were shown to express the CD68 marker, suggesting that infected mononuclear phagocytes crossing the blood-brain barrier represent the main source of virus in the CNS. Early viral replication coincided with neuropathologic changes, consisting in gliosis, perivascular infiltrates and rare glial nodules. Immunophenotyping of brain tissue showed that increased macrophage infiltration, microglial reactivity and MHC class II induction occurred within the first week of infection, indicating a possible immunopathologic mechanism in early CNS pathogenesis.

Virus load and neuropathology in the FIV model.

The FIV (feline immunodeficiency virus) induces in cats brain changes presenting similarities with those observed in human immunodeficiency virus infection. This FIV model was used to study the relationship between viral load in brain, in lymphoid organs and central nervous system (CNS) changes during the early and late stages of infection. Early brain changes were analyzed in animals experimentally infected with two different FIV isolates and sacrificed at 7 and 15 days, 1, 2, 6, and 12 months post inoculation (p.i.). Late CNS abnormalities were analyzed in naturally FIV-infected cats referred to the Veterinary School of Nantes. For each animal, one cerebral hemisphere was fixed and examined using routine techniques. The characterization of FIV replicating cells by in situ hybridization was performed on the other half frozen hemisphere on sections performed in the anterior and the median regions of the brain. During the early stages of infection, moderate gliosis with glial nodules and sometimes white matter pallor and meningitis were associated with few infected cells scattered in the brain. Infection was an early event as infected cells could be detected in brain at 7 p.i. For each cat, these findings were found identical in the two analyzed areas. During the late stages, brain lesions and the number of virus replicating cells increased especially in animals with perivascular infiltrates. The multinucleated giant cells encephalitis was never observed and the number of FIV replicating cells scattered in the whole brain was always low. This discrepancy between the number of replicating cells and the brain lesions, corroborates the hypotheses suggesting that brain injuries may be mediated via diffusive factors and amplification processes through cytokine cascades and cell activations.

Early stages of simian immunodeficiency virus infection in lymph nodes. Evidence for high viral load and successive populations of target cells.

Lymph nodes obtained from 14 macaques sacrificed at early time points following experimental inoculation with simian immunodeficiency virus were analyzed by in situ hybridization for virus load and virus cellular tropism. The lymph nodes presented a remarkably high viral load during the early phase of infection, as viral RNA was detected in as many as 2% of lymph node cells 1 week after inoculation. At this stage, macrophages and T4 lymphocytes were identified by combined immunohistochemistry and in situ hybridization as the target cells of the virus. Simian immunodeficiency virus-positive macrophages concentrated in the subcapsular sinuses, suggesting an entry of infected cells via the afferent lymphatics. A shift in the pattern of viral infection was observed at 2 weeks after inoculation, with a concentration of viral RNA in the germinal centers of the developing lymphoid follicles. Follicular dendritic cells were found to be the major target of the virus at this stage. Follicular dendritic cells were associated with high levels of viral RNA but little or no detectable viral DNA, suggesting that the virus was present mostly in the form of viral particles trapped at the cell surface. Follicular dendritic cell-associated virus persisted at high levels for 2 months before subsiding, indicating that follicular dendritic cells constituted a major reservoir of the virus during the early stages of simian immunodeficiency virus infection.