Polybrominated diphenyl ethers and polychlorinated biphenyls in human breast adipose samples from Brazil.

Twenty five human breast adipose tissue samples were collected in Porto Alegre, Brazil during 2004-2005 and analyzed for polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs). SigmaPBDE concentrations (sum of tri- to hepta-BDEs) ranged from 0.19 to 132 ng/g lipid with a median of 1.51 ng/g lipid. These concentrations are 3- to 100-times lower than those reported from other countries, with the exception of Japan, probably reflecting lower usage of PBDE-containing products or lower exposures to these chemicals. The predominant congener was BDE-47, followed by BDEs 99, 183, 153 and 100. One individual in the dataset had about 70-times higher PBDE concentrations than the rest of the participants. SigmaPCB (sum of PCBs 118, 138, 153, 180) ranged from 30 to 339 ng/g lipid, with a median of 51 ng/g lipid. No age dependency was found for PBDEs (r=-0.800-0.374, p>0.05) or PCB 180 (r=0.278, p>0.05). On the other hand, PCBs 118, 138 and 153 did show age dependency (r=0.410-0.458, p<0.05). This is the first study to report levels of PBDEs in human breast adipose from Brazil.

Interindividual variation in CYP1A1 expression in breast tissue and the role of genetic polymorphism.

The cytochrome P4501A1 (CYP1A1) enzyme is regulated at the transcriptional level and its expression is influenced by genetic factors, polymorphisms in the structural and regulatory genes, and by environmental factors such as exposure to polycyclic aromatic hydrocarbons (PAHs). To investigate the role of CYP1A1 in breast cancer, we studied CYP1A1 expression in breast tissue, thereby taking all possible modifying factors into account. We measured CYP1A1 expression in 58 non-tumor breast tissue specimens from both breast cancer patients (n = 26) and cancer-free individuals (n = 32) using a newly developed reverse transcription-polymerase chain reaction assay. CYP1A1 expression varied between specimens approximately 400-fold and was independent of age. CYP1A1 expression was somewhat higher in tissue from breast cancer patients than in that from cancer-free individuals, but this difference was not statistically significant. Analysis for CYP1A1 genetic polymorphisms revealed eight variants, seven in the cancer-free group and one in the patient group. The variant genotype was not a good predictor of expression level. We conclude that high CYP1A1 expression could be a risk factor for breast cancer and that the known CYP1A1 polymorphisms are not good predictors of CYP1A1 expression.

Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT.

Studies were carried out to determine whether apolipoprotein (apo) A-II, like apoA-I, can recruit phospholipid and cholesterol from cell membranes, thereby forming nascent apoA-II-specific HDL. ApoA-II and apoA-I were purified from plasma and each was incubated with CHO cells at a concentration of 10 micrograms/ml. Lipid-containing complexes were isolated from the medium in both cases; the composition of the apoA-II- and apoA-I-specific complexes were similar where percent protein, phospholipid, and cholesterol were 35 +/- 3, 38 +/- 2, and 25 +/- 1 for apoA-II, respectively, and 40 +/- 2, 35 +/- 1, and 24 +/- 2 for apoA-I, respectively. On a per mole of apolipoprotein basis, apoA-I recruited significantly more phospholipid and cholesterol than dimeric apoA-II suggesting that apoA-I with its greater number of alpha helices binds more lipid. By electron microscopy, nascent apoA-II- and apoA-I-specific particles were predominantly discoidal in morphology. ApoA-II complexes were unique in their nondenaturing polyacrylamide gradient gel size distribution as six distinct populations of particles with diameters of 8.1, 9.3, 10.4, 11.8, 13.1, and 14.6 nm were routinely noted, compared with apoA-I which formed only three major populations with diameters of 7.3, 9.2, and 11.0 nm. Nascent apoA-I complexes incubated with purified lecithin:cholesterol acyltransferase (LCAT) were transformed into predominantly 8.4 nm particles. The latter is similar in size to plasma HDL3a, LpA-I particles, suggesting that extracellularly assembled apoA-I-lipid complexes can directly give rise to a major plasma LpA-I subpopulation upon interaction with LCAT. Unlike apoA-I, apoA-II-lipid complexes could not serve as substrates for LCAT and did not undergo transformation. This study also demonstrates, for the first time, that apoA-II and apoA-I show a preference in phospholipid recruitment from membranes. Although phosphatidylcholine is the major phospholipid removed by both apolipoproteins, apoA-II preferentially recruits phosphatidylethanolamine (PE) as its second most abundant phospholipid while apoA-I recruits sphingomyelin. As PE is usually associated with the inner leaflet of the membrane, it is likely that dimeric apoA-II, compared with apoA-I, can penetrate farther into the membrane and extract PE. This ability of apoA-II to insert more deeply into the lipid milieu may explain the known ability of apoA-II to resist dissociation from the mature HDL particle.

Apolipoprotein A-I-cell membrane interaction: extracellular assembly of heterogeneous nascent HDL particles.

The ability of lipid-free human apoA-I expressed by transfected Chinese hamster ovary (CHO) cells to form apoA-I-lipid complexes extracellularly when incubated with CHO cell monolayers was investigated. Lipid-free apoA-I was incubated with nontransfected CHO-C19 cells for 24 h and extracellular assembly products were isolated at d < or = 1.235 g/ml; approximately 12% of the incubated apoA-I floated at d < or = 1.235 g/ml when apoA-I was added at 10 micrograms/ml. The composition of the particles was 51.3% protein, 20.3% phospholipid, and 28.3% cholesterol. Electron microscopy of the apoA-I-lipid complexes revealed that discoidal particles 15.4 +/- 4.1 nm diameter predominated but some vesicular particles 34.7 +/- 16.8 nm diameter were also in evidence. Nondenaturing gradient gel electrophoresis of the extracellular assembly products formed after 24 h incubation with 10 micrograms/ml apoA-I showed particle size heterogeneity with major bands at 11.2 and 9.0 nm; additional minor components banded at 7.3, 17.7, and 19.5 nm. This size distribution, as well as composition and electron microscopic structure, is similar to that of complexes isolated from the medium of CHO cells transfected with the human apoA-I gene. The formation of extracellular assembly complexes was concentration-dependent such that at 2 micrograms apoA-I/ml for 24 h, primarily 7.3 nm complexes were formed; at 4 micrograms/ml the distribution was more heterogeneous and the major band peaked at 9.2 nm, while at 8 micrograms/ml the 7.3 nm component was greatly diminished and the 11.2 nm component was the major one.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair of alkylation damage in Saccharomyces cerevisiae.

Repair of methylated bases in Saccharomyces cerevisiae was measured by two methods: in vitro in cell extracts, and in vivo, by determining the loss of methylated bases from yeast DNA after treatment of stationary cultures with [3H]-N-methyl-N'-nitro-N-nitrosoguanidine. Whereas no repair activity could be detected by the in vitro method, the methylated bases were removed in vivo very efficiently. These contradictory results of in vitro and in vivo repair measurements suggest that either the repair enzymes of yeast are sufficiently different from those of bacteria and mammalian cells that they are not active in the in vitro assay, or that methylated bases are repaired in yeast by a different pathway.

MNNG-induced partial phenotypic reversion of Mer- cells.

The effect of pretreatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on MNNG sensitivity of the surviving population was compared in two HeLa lines, one of the Mer+ phenotype (HeLa S3) and one of the Mer- phenotype (HeLa MR). Whereas MNNG pretreatment of HeLa Mer+ cells had no effect on the MNNG sensitivity of surviving cells, Mer- cells surviving a first exposure to MNNG became much more resistant to MNNG. Comparison of the sensitivity of individual HeLa MR clones with their MNNG-pretreated population and analysis of the composition of the pretreated population showed that the majority of cells surviving the MNNG pretreatment now displayed the Mer+ phenotype in respect to sensitivity to MNNG. One MNNG-resistant clone derived from a pretreated HeLa MR population (Cl 4) was characterized further. It had a similar sensitivity to the Mer+ line to all monofunctional alkylating agents, but was as sensitive as the Mer- line to the crosslinking agent chloroethylnitrosourea. Cl 4 cells, like the Mer- cells, did not repair O6-methylguanine (O6MeG). The results suggest that the two characteristics which are usually coupled with the Mer- phenotype--lack of O6MeG repair and hypersensitivity to MNNG--can be separated.

Characterization of a CHO variant in respect to alkylating agent-induced biological effects and DNA repair.

From the Chinese hamster ovary line CHO-9 a resistant variant, Cl 3, was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cl 3 cells were much more resistant to the cytotoxic effects of MNNG (D10 of 1.8 microgram/ml MNNG as compared to 0.23 microgram/ml for parental line) and other methylating N-nitroso compounds, but they had the same sensitivity to various other alkylating agents. MNNG was equally effective in sensitive parent line and resistant variant in inducing sister-chromatid exchanges (SCEs) and mutations to 6-thioguanine resistance. The increased resistance of Cl 3 was not due to reduced cellular uptake of MNNG, to a more efficient repair of methylated purine bases, or to differences in MNNG-induced inhibition of DNA synthesis. It is concluded that the resistant variant has some unknown tolerance mechanism which alters the cytotoxic, but not the SCE- and mutation-inducing effects of methylating N-nitroso compounds.

Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells.

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.

Toxicity of 4-nitroquinoline 1-oxide in Chinese hamster ovary cells: influence of cell density and of position in the cell cycle.

Various factors influencing toxicity of 4-nitroquinoline 1-oxide (4-NQO) in Chinese hamster ovary cells were determined. Cell density during 4-NQO treatment and volume of treatment medium had a great effect on cell survival indicating that not the 4-NQO concentration per se, but the amount of 4-NQO per cell determines the toxic effect. When the cell-cycle response for 4-NQO-induced cell killing was measured in synchronous cells, a characteristic age response was seen in wild-type cells with greatly increased sensitivity in late G1 to early S and resistance increasing through the S-phase. In contrast, a UV-hypersensitive mutant, which is also more sensitive to 4-NQO showed only minor cell-cycle variations in its response to 4-NQO. Therefore, it appears that the cell-cycle pattern observed in the wild-type cells is associated with DNA repair.

Cell killing by various nitrosoureas and the potentiating effect of caffeine.

Cell killing by various nitrosoureas and the potentiating effect of caffeine on cell killing were quantified by means of colony-forming assays in a clonal derivative of M3-1 Chinese hamster cells. The dose-response relationships for ethylnitrosourea and 3 chloroethylnitrosourea derivatives were compared with that of 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea, which is a purely carbamoylating agent. The chloroethylnitrosoureas with both alkylating and carbamoylating activity were the most toxic, followed by a chloroethylnitrosourea with alkylating activity alone and then by the purely carbamoylating agent. The least toxic agent was ethylnitrosourea, which has both alkylating and carbamoylating activity. Caffeine (when present through the entire period of colony growth) potentiated cell killing for all nitrosoureas with alkylating activity but had no influence on cell killing by the purely carbamoylating agent. Because carbamoylation occurs mainly with proteins, whereas nucleic acids as well as proteins are cellular targets for alkylation, it is concluded that potentiation of cell killing by caffeine is based on the reaction of a drug with the cellular DNA.

Effect of caffeine on cell killing, mutation induction, DNA repair, and DNA synthesis after treatment with ethylnitrosourea.

The effect of caffeine on cell killing, mutation induction, DNA repair, and inhibition of DNA synthesis was investigated in a clonal derivative of M3-1 Chinese hamster cells after treatment with N-ethyl-N-nitrosourea (ENU). Caffeine enhanced cell killing but had no effect on the mutation frequency/viable cells for the two genetic markers, 6-thioguanine resistance and ouabain resistance. The removal of ethylated purine bases from DNA was as follows: most of the 3-ethyladenine was lost in 20 h (greater than 85%) and approximately 45% of the 7-ethylguanine was lost in 45 h, whereas 75--93% of the O6-ethylguanine was still present at this time. Caffeine did not seem to influence these rates significantly. The ENU-induced inhibition of DNA synthesis was reversed by caffeine. It is concluded that the potentiation of ENU-induced cell killing by caffeine is caused by the increased frequency of DNA replication past damaged sites in parental DNA.

Inability of Chinese hamster ovary cells to excise O6-alkylguanine.

The capacity of Chinese hamster ovary cells for excising alkylated bases from their DNA was investigated. Cells were treated with N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea, and the amounts of 3-alkyladenine, 7-alkylguanine, and O6-alkylguanine in DNA were compared immediately after the treatment and after a 20-hr incubation. Cells were able to excise 3-alkyladenine and 7-alkylguanine, but there was no loss of O6-alkylguanine, after correction for dilution, due to DNA synthesis during posttreatment incubation. The inability of Chinese hamster ovary cells fail to show growing point mutagenesis after treatment with N-ethyl-N-nitrosourea.

Ethylnitrosourea-induced mutagenesis in asynchronous and synchronous Chinese hamster ovary cells.

The toxic and mutagenic activity of the alkylating carcinogen N-ethyl-N-nitrosourea (ENU) was studied in Chinese hamster ovary (CHO) cells. Cell killing and induction of 6-thioguanine-resistant (TGr) and ouabain-resistant (OUAr) mutants were determined as a function of ENU dose and treatment time in asynchronous cell populations. A dose-dependent induction of mutants was observed. The mutation frequency did not increase with longer than 30-min treatment times, implying that ENU breaks down rapidly in the cell. When synchronous populations of CHO cells obtained by mitotic detachment were treated with ENU at various times during the cell cycle, ENU-induced reproductive death was strongly dependent on the position in the cell cycle at the time of treatment, the time of highest sensitivity being the beginning of the S period. The pattern of mutation induction by ENU over the cell cycle was quite different from the pattern for cell killing. The induction of TGr mutants seemed to be independent of cell-cycle time. The induction of OUAr mutants was also independent of cell-cycle time after a low ENU dose; however, after a high ENU dose the frequency of OUAr mutants varied during the cell cycle, with a slight enhancement in B1 and a decrease in the early S period. There was no sign of enhanced mutation induction at the growing point for the two genetic markers tested.