Intraperitoneal linear polyethylenimine (L-PEI)-mediated gene delivery to ovarian carcinoma nodes in mice.

Linear polyethylenimine (L-PEI) is an efficient transfection agent for ovarian carcinoma cells in vitro and ex vivo. In the present work, we go a step further and evaluate the efficacy of L-PEI in human ovarian tumor nodes developed in mice. PEI/DNA complexes were administered intraperitoneally instead of intravenously to avoid sequestering of complexes in the lung and liver and to allow transfection of nonvascularized tumor nodes. Plasmid biodistribution was studied by PCR and gene expression was characterized using complementary luciferase and beta-galactosidase assays. Intraperitoneal (i.p.) injection of L-PEI/DNA complexes allowed the straightforward distribution of plasmid in the whole peritoneal cavity. Gene expression occurred in many organs, but tumor nodes appeared as preferential sites for transgene expression. The i.p. delivery route allowed repeated injections and administration of large amounts of DNA (up to 400 mug) without signs of toxicity, even for doses well beyond the intravenous lethal dose. Transgene expression was dose-dependent and transient. However, multiple injections allowed its persistence to increase. These results provide encouraging elements towards the development of PEI-based gene therapy protocols for the treatment of advanced stage ovarian carcinoma.

Clinical value of serum glycoprotein galactosyltransferase levels in different histological types of ovarian carcinoma.

Serum glycoprotein galactosyltransferase levels were determined in 28 healthy women and 113 patients with ovarian carcinoma with various histological types, at different clinical stages. Ovomucoid, which possesses terminal N-acetylglucosaminyl residues, was used as glycoprotein acceptor. Clinical correlations between galactosyltransferase levels and tumor burden were examined, as well as the variations due to histology. Follow-up studies could be done for 60 patients, and correlations with clinical evolution, established. Galactosyltransferase might be a promising marker for the diagnosis and follow-up of ovarian carcinomas.

Seasonal variations of DNA-adduct patterns in open field farmers handling pesticides.

Seasonal variations of DNA-adduct levels in peripheral blood cells were evaluated in open field farmers (n=26) by use of the 32P-postlabelling assay. Samples were collected before (sample S0) and during (sample S4) the period of intensive pesticide use. A similar sampling procedure was applied to a referent group (n=29). Exposure to pesticides was estimated via a detailed questionnaire. For the group of farmers, an increase in mean adduct level was observed during the season (mean RALS0=3.9+/-3.4 x 10(-10), mean RALS4=13.3+/-15.7 x 10(-10); p=0.008; RAL=relative adduct level). The mean adduct levels were significantly different between farmers and referents only in the S4 samples, with higher levels for farmers (p=0.02). The number of different adducts per individual was higher for farmers at S4 when compared with S0 (p=0.02) and compared with the referents at S4 (p=0.03). However, the increase of the adduct level in farmers did not seem to be attributable to the occurrence of specific new adducts in S4 as compared with S0, but was supposedly due to intensification of pre-existing spots and/or appearance of new unspecific ones. This would be in agreement with indirect genotoxic (epigenetic) effects known for several pesticides, even though a direct mechanism cannot be ruled out definitively. The implication of the pesticides used by the farmers in the modulation of DNA-adduct patterns was explored by analysis of exposure data obtained from the questionnaire.

DNA damage in mononuclear leukocytes of farmers measured using the alkaline comet assay: discussion of critical parameters and evaluation of seasonal variations in relation to pesticide exposure.

The alkaline comet assay was used to quantify, using visual and image analyses, the level of DNA damage in mononuclear leukocytes of farmers who were occupationally exposed to pesticides. Hematological parameters were also measured on the same samples. Enrollment of farmers was based on handling of heavily used pesticides at particular periods during one spraying season. Forty-one blood samples from 29 different farmers were collected at the beginning of the season (n = 11) and at the intermediate (n = 14) and final (n = 16) periods of intense spraying activity. The mean numbers of lymphocytes and eosinophils were nonsignificantly higher in groups 3, 1, and 4 than they were in group 2. No individual characteristics significantly influenced the mean number of lymphocytes or eosinophils, and no correlation was observed between pesticide exposure-related parameters and hematological parameters. The level of DNA damage was significantly (P < 0.01) higher in groups 3, 1, and 4 than it was in group 2. In addition, DNA damage quantification was not significantly different among investigators or among slides. Prescription medicine, alcohol consumption, and age had no statistically significant effect on DNA damage level. Conversely, smoking (smokers versus non- and ex-smokers) significantly influenced DNA damage level (P < 0.0001). A significant (P < 0.05) negative correlation was detected between the number of days without pesticide spraying and DNA damage level, particularly among non- and ex-smokers. DNA damage detected by the alkaline comet assay seems to reflect ongoing exposure to genotoxic agents but not an accumulation of damage.

DNA damage in mononuclear leukocytes of farmers measured using the alkaline comet assay: modifications of DNA damage levels after a one-day field spraying period with selected pesticides.

The alkaline comet assay was used to assess DNA damage in mononuclear leukocytes of farmers before and after a 1-day spraying period with selected pesticides under usual conditions. Two blood samples were collected, one in the morning of the day of spraying (S0) and the second in the morning of the day after (S1). Here, we assessed variations in DNA damage levels between these two sampling times. Four groups of farmers were formed, according to exposure to: (a) various fungicide-insecticide mixtures (including chlorothalonil; group 1, n = 8), (b) the herbicide isoproturon (group 2, n = 11), (c) fungicide triazoles (group 3, n = 14), and (d) a fungicide (chlorothalonil)-insecticide mixture (group 4, n = 8). An increase in DNA damage levels was observed at S1 for groups 1 and 4, who were exposed to similar pesticides. This increase was correlated with area sprayed between S0 and S1 and with the number of spraying tanks used over this 1-day period. No effect was observed on cell viability or on hematological parameters for these two groups. No statistically significant modification of DNA damage level was observed the day after spraying for groups 2 and 3, when each was observed as a whole. However, some farmers presented significantly more DNA damage after exposure, and others presented less damage. In these two groups, a significant decrease of neutrophils was observed at S1, and a decrease of red blood cells was observed in group 3. In parallel, a significant loss of lymphocyte viability was observed in these two groups. A 1-day spraying period seems to be sufficient to significantly modify DNA damage levels in mononuclear leukocytes, but the correlation of this change with pesticide-related exposure parameters depends on the kind of pesticide concerned.

Charge heterogeneity of human mammary tumor estrogen receptors. Relationship with a hormonal sensitivity tumor marker.

A high resolution quantitative method for estrogen receptor analysis has been elaborated using isoelectric focusing in 0.5% agarose gel, without any prior trypsin digestion. The 23 cytosols analyzed were stabilized by molybdate and prepared from human mammary tumors with progesterone receptors (PR + cytosols) or without (PR - cytosols). Progesterone receptor was used as a tumoral hormonal sensitivity marker. The estrogen receptors usually resolved as 4 isoform peaks of close isoelectric points. In PR - cytosols, the mean pI values were 4.7, 5.5, 6 and 6.5. Significant differences between the two cytosol populations were observed concerning pI 4.7 and 6.5 isoforms. In PR - cytosols, the pI 4.7 isoform occurred in greater proportions than in PR + cytosols, whereas lower proportions of pI 6.5 isoform were seen. The comparison between high performance size exclusion chromatography profiles and isoelectric focusing patterns, before and after cytosol incubation at 28 degrees C with KCl (0.4 M), confirmed an oligomer structure for the pI 4.7 isoform and suggested a monomer structure (Stokes radius 2.9 mm) for the pI 6.5 estrogen receptor isoform. The results indicated that isoelectric focusing analysis of estrogen receptors could be useful in the prediction of breast cancer hormonal sensitivity.

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells.

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21(cip1/waf1) on ovarian carcinoma chemosensitivity. The transfection of p21(cip1/waf1) cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21(cip1/waf1) gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21(cip1/waf1) as an adjunctive to conventional chemotherapy.

No BCL-2 protein over expression but BCL-2/IgH rearrangements in B cells of patients with persistent polyclonal B-cell lymphocytosis.

INTRODUCTION: Persistent polyclonal B-cell lymphocytosis is a rare hematological disorder, characterized by a chronic, stable and absolute polyclonal lymphocytosis, the presence of binucleated lymphocytes, a polyclonal increase in serum IgM immunoglobulin and clonal cytogenetic abnormalities involving chromosome 3. For explaining the expansion of B-lymphocytes pool in PPBL, an association with cigarette smoking and/or chronic Epstein-Barr virus infection have been suggested but both hypotheses have been ruled out. MATERIALS AND METHODS: We studied the presence of BCL-2/IgH rearrangements in a series of eight PPBL patients (seven females and one male) by a nested polymerase chain reaction (PCR), targeting the Major Breakpoint Region in BCL-2 locus and we explored the BCL-2 protein expression by Western blot. RESULTS: We demonstrated: (a) the constant presence of BCL-2/IgH rearrangements in eight out of eight DNA samples, (b) multiple rearrangements in three out of eight cases and, (c) normal BCL-2 protein expression, as compared to BCL-2 level in B-lymphocytes from healthy population. CONCLUSION: Despite the presence of BCL-2/IgH rearrangements, the accumulation of B lymphocytes in PPBL is not related to an overexpression of BCL-2 protein.

Potentiation of cisplatin and carboplatin cytotoxicity by amphotericin B in different human ovarian carcinoma and malignant peritoneal mesothelioma cells.

An in vitro study of the combined cytotoxicity of either cisplatin (CDDP) or carboplatin and amphotericin B (AmB) was undertaken on a set of different ovarian carcinoma (IGROVI, IGROVI-C10, OAW42) and peritoneal malignant mesothelioma (CFB-CARP1) cell lines and ascitic cells freshly obtained from ovarian cancer patients so as to investigate the possibility of overcoming their resistance to platinum compounds. Growth-inhibition curves obtained 6 days after a 2-h period of exposure to the drugs showed that AmB at 5-10 mg/l allowed a 5- to 10-fold decrease in the 50% growth-inhibitory concentrations (IC50) of CDDP and carboplatin on either sensitive or resistant cells. Intracellular platinum assays with IGROVI cells showed that AmB acted by increasing dramatically the platinum uptake at a proportion that accounted for the increase in cytotoxicity. In the subline IGROVI-C10, a 10-fold resistant subline of IGROVI, AmB at 10 mg/l allowed recovery to the level of sensitivity seen in the parental cell line in the absence of AmB but not to the level observed in the presence of AmB. Acquisition of resistance mechanisms that are independent of the regulation of platinum uptake might be involved in this cell line. Thus, AmB might act by increasing the intracellular concentration of platinum without modifying the resistance mechanism involved downstream. However, in our models an increase in the intracellular level of platinum was always sufficient for the recovery of chemosensitivity in vitro. We also show that the phosphodiesterase inhibiting methylxanthines act synergistically with AmB. The latter drugs are weakly toxic and could also attenuate the nephrotoxicity of AmB.

A simple serum galactosyltransferase assay method suitable for routine use.

UDP-Galactose: N-acetylglucosaminyl glycoprotein beta 1-4 galactosyltransferase (GT) catalyzes the transfer of galactose to N-acetylglucosamine from UDP-[3H]Gal. The uncharged reaction product (tritiated N-acetyllactosamine) is separated from the unreacted UDP-[3H]Gal by ion-exchange chromatography. The major advantage of this method is its rapidity compared to other isotopic techniques.

Biological markers and ovarian carcinomas: galactosyltransferase, CA 125, isoenzymes of amylase and alkaline phosphatase.

We present a comparative study of several biological markers (galactosyltransferase, CA 125, isoenzymes of amylase and alkaline phosphatase) with a view to ovarian carcinoma follow-up. Serum samples were obtained from a population of 75 patients under clinical observation. After a minimum 18-months period, we assessed the prognostic value of the markers. No marker permits the detection of discrete, evolving carcinomas. CA 125 is the marker that gives the best results, particularly in terms of sensitivity. Galactosyltransferase has a lower sensitivity except in the case of endometrioid carcinomas. Simultaneous analysis with CA 125 and galactosyltransferase results in no decisive improvement, other than greater precision in unfavourable prognoses. Isoenzymes of amylase and alkaline phosphatase are of no interest in the follow-up of such carcinomas.

Pulse fluorometry of cyclo-(glycyl-L-tryptophyl).

The fluorescence of cyclo-(glycyl-L-tryptophyl) in trimethyl phosphate has been studied in a temperature range varying from room temperature to -85 degrees C. At room temperature, the fluorescence decay is the sum of two exponentials, the relative amplitude of which depends on the emission wavelength. This can be explained by the presence of the two following emitting molecular states: on one hand the unfolded state, the fluorescence decay time and the emission spectrum of which are close to these of skatole; on the other hand the folded state which has a shorter decay time and a blue-shifted spectrum. By lowering the temperature, the fluorescence spectrum shifts to the blue, while the skatole spectrum shifts to the red. This behavior corresponds to an increase of the folded conformation concentration in agreement with the NMR results. Furthermore the rate of exchange between the folded and the unfolded conformations decreases. Accordingly the wavelength dependence of the fluorescence decay lessens. There are two possible values of the conformational angle x2 differing by 180 degrees, which correspond to the folded state; due to the indole asymmetry, the interactions between the indole and diketopiperazine rings differ in these conformers. Consequently the fluorescence decay remains biexponential even at -85 degrees C.

Alteration in isoenzyme patterns of serum galactosyltransferase activity in ovarian cancer patients: preliminary results.

Isoelectric focusing on agarose gel was used to separate the isoenzymes of serum galactosyltransferase (uridine diphosphogalactose: N-acetylglucosaminyl galactosyltransferase, EC 2.4.1.22) from 8 healthy women, and 11 ovarian cancer patients of whom 4 were in clinical remission. In all cases, we found 7 major peaks with isoelectric points ranging from 4.0-5.4. The most acidic peaks were preferentially elevated in the tumor-bearing patients, particularly the peak with pI 4.44.

Urine mutagenicity and lymphocyte DNA damage in fruit growers occupationally exposed to the fungicide captan.

AIMS: To determine haematological parameters, urine mutagenicity (on three Salmonella typhimurium strains), and DNA damage (using the comet assay) in mononuclear leucocytes of farmers before and after a one-day spraying period of pear and apple trees with the fungicide captan in usual conditions. METHODS: Fruit growers were exposed to captan during the 1998 (n = 12) and/or the 2000 spraying seasons (n = 17). Biological samples were collected on the morning of the day of spraying (S1), the evening after spraying (S2), and the morning of the day after (S3). The UK Predictive Operator Exposure Model (UK-POEM) was used to quantify pesticide exposure intensity. RESULTS: No effect was observed on haematological parameters for these two spraying seasons. Proportions of mutagenic urine samples did not significantly differ between S1 and S2/S3 sampling points. In contrast with strains TA97a and YG1041 mainly sensitive to frameshift mutations, a positive trend was observed between the difference (S3-S1) of mutagenic power on strain TA102 detecting base-pair mutations and the exposure predicted value given by UK-POEM, mainly due to parameters related to protective clothing. No significant variations in DNA damage levels were observed between S1 and S3, nor were correlations observed with parameters of pesticide exposure. CONCLUSIONS: A one-day spraying period with captan and other pesticides does not significantly induce DNA damages in mononuclear leucocytes. In contrast, an inefficient protective clothing could correlate with an increase in urine mutagenicity as assessed by the TA102 tester strain.

Modulatory effects of melatonin on genotoxic response of reference mutagens in the Ames test and the comet assay.

The effect of a potent endogenous antioxidant, the pineal gland indole melatonin (MLT) on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay (SCGE assay or COMET assay). The 12 mutagens used were 7, 12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene (AF), 1,2-dimethylhydrazine (DMH), bleomycin, cyclophosphamide (CP), 4-nitroquinoline-N-oxide (NQO), 2,4, 7-trinitro-9-fluorenone (TNF), 9-aminoacridine (AA), N-nitrosomethylurea (NMU), mitomycin C and sodium azide tested in the absence or in the presence of S9 mix. MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 microM) in the SCGE assay. In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation. In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU. With mitomycin C, MLT exacerbated responses in both tests. The possible mechanisms of MLT's inhibitory action are discussed.

Mutagenicity of nitro- and amino-substituted carbazoles in Salmonella typhimurium. I. Monosubstituted derivatives of 9H-carbazole.

Mononitro, monoamino and monoacetamido carbazoles were assayed for mutagenicity in Salmonella typhimurium strains TA1535, TA1538, TA1537, TA1977, TA98 and TA100, with and without the addition of S9 from phenobarbital-induced rat liver. The role of bacterial metabolism of the nitro group was also studied using the additional strains TA98NR and TA98/1,8DNP6. None of the compounds was active in TA1535, while only 2-nitrocarbazole and 3-nitrocarbazole presented a weak mutagenicity towards its pKM101 derivative, TA100. All four nitrocarbazole isomers were mutagenic for TA1538 and TA98, their activities decreasing in the order: 2-nitrocarbazole approximately 3-nitrocarbazole > 1-nitrocarbazole > 4-nitrocarbazole. Direct-acting mutagenicities for TA1537 were lower than for TA1538, but varied in the same order. Nitro reduction was an important step of metabolic activation of nitrocarbazoles, as indicated by the dramatic reduction of activity with TA98NR, as compared to TA98. Results obtained with TA98/1,8DNP6 showed that O-acetyltransferase was only partly required for the expression of mutagenic potency of these compounds. 2-Aminocarbazole was a weak direct-acting mutagen for TA1538 and TA98. Its activity was strongly increased in the presence of S9 mix, while 3-aminocarbazole became active in these conditions. The acetamido derivatives were consistently less mutagenic than their parent amines. These results show that nitrocarbazoles and aminocarbazoles behave as reactive frameshift mutagens, acting mainly through the formation of esterified hydroxylamines. The very low activity of 4-nitrocarbazole might be related to an orientation of the nitro group perpendicular to the aromatic ring.

Mutagenicity of nitro- and amino-substituted carbazoles in Salmonella typhimurium. III. Methylated derivatives of 9H-carbazole.

The mutagenic potency of nine methylnitrocarbazoles, four methylaminocarbazoles and the methylcarbazole parent compounds was evaluated in Salmonella typhimurium TA98 and TA100, in the absence and presence of S9 isolated from Aroclor-induced rat liver. Nitro derivatives were additionally tested in TA98NR and TA98/1,8DNP6, and mutagenicity of nitrocarbazoles bearing methyl groups in positions 1 and 4 was also determined in TA1537 and TA1977, with and without S9. The addition of methyl groups on non-mutagenic carbazole can induce a mutagenic response where the intensity and nature of the effect depends on the position of the substitution: base-pair substitutions were only observed for N-methylated carbazoles, whereas 1,4-dimethylated compounds exhibited frameshift mutagenicity. All these activities depended on the presence of S9. From its dependence on classical nitroreductases, direct mutagenicity of methylnitro derivatives should be attributed to bacterial reduction of nitro groups. The influence of the methyl groups (and other additional substituents) on mutagenicity of these derivatives is discussed through their effects on life-time and reactivity of the intermediates (i.e., hydroxylamines and nitrenium ions), taking into account the nature, the position and the number of substituted sites Mutagenic activity of methylnitrocarbazoles was also tentatively correlated with various molecular descriptors. Among them hydrophobicity was found to be strongly correlated with the mutagenicity of the 1,4diMe3NC isomers. On the other hand, mutagenic potency of the nitrated and aminated methylcarbazoles varied independently of parameters linked to their oxidoreduction properties (i.e., reduction and oxidation potentials, LUMO and HOMO energies).

Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorothalonil) in human lymphocytes with the comet assay.

The effects of two fungicides (carbendazim and chlorothalonil) on the induction of DNA damage in human peripheral blood lymphocytes (human PBL) have been investigated using the single cell gel electrophoresis assay (SCGE assay or comet assay) immediately after a 1-h treatment and after a 24-h post-treatment incubation. The assessment of etoposide (an effective antitumour agent) effects on human PBL in terms of cell viability and dose-DNA damage relationships was made and etoposide selected as a positive control. The results indicate that etoposide induces significant (p < 0.01) dose-dependent DNA damages for concentrations at which the loss of cell viability is low. After a 24-h recuperation period, all observed DNA damages has disappeared. With SCGE assay performed after a 1-h treatment, similar positive results were observed with chlorothalonil alone or in association with carbendazim, without any loss of cell viability. However, a dramatic loss of cell viability was measured after 24 h and was associated with a large proportion of highly damaged cells. In contrast, carbendazim was not cytotoxic on human PBL and did not induced DNA damage using the SCGE assay either immediately after treatment or after a 24-h post-treatment incubation. These results point to the necessity of an adequate evaluation of immediate and long-term cytotoxicity of compounds that are to be assessed by the SCGE assay.

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells.

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.