Tolerance to O6-methylguanine and 6-thioguanine cytotoxic effects: a cross-resistant phenotype in N-methylnitrosourea-resistant Chinese hamster ovary cells.

The biochemical and genetic characteristics of a clone of Chinese hamster ovary cells displaying resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (6-TG) were analyzed. The initial level of 7-methylguanine, 3-methyladenine, and O6-methylguanine formation and the repair rates for these alkylated bases were the same in the resistant and in the parental cell line, indicating that the resistance to alkylation damage is not due to differences in DNA alkylation. After exposure for 24 or 48 h to 6-TG (0.6 micrograms/ml) in culture medium, the resistant clone in contrast to them, was able to replicate the DNA containing the base analogue during the following 24 h. These data are in agreement with the hypothesis that resistant cells tolerate both O6-methylguanine and 6-TG present in DNA. The tolerance to MNU and 6-TG also included chromosomal damage induced by these two agents, and MNU-resistant cells incurred less sister chromatid exchanges after treatment with either MNU or 6-TG. 6-TG-resistant cells, selected by growth in 6-TG, exhibited cross-resistance to MNU but not to methyl methanesulfonate, confirming that a common pathway of tolerance is responsible for resistance to 6-TG and O6-methylguanine.

Quantitative relationship between ethylated DNA bases and gene mutation at two loci in CHO cells.

In a previous study we showed that the formation of O6-ethylguanine (O6-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N7 and O6 positions of guanine and at the N3 site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TGr) and ouabain resistance (ouar) mutations were investigated. A good correlation between the levels of ethylation at O6 guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the ouar locus, the frequency of O6-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multicomponent phenomena like mutation formation.

Clean-up of aqueous acetone vegetable extracts by solid-matrix partition for pyrethroid residue determination by gas chromatography-electron-capture detection.

Disposable, ready-to-use cartridges filled with macroporous diatomaceous material are used to carry out a partition clean-up that, in a single step, is capable of transferring pesticide residues from aqueous acetone extracts into light petroleum-dichloromethane (75:25, v/v). This procedure takes the place of some functions (such as separatory-funnel partition, drying over anhydrous sodium sulphate and partial adsorption clean-up) usually performed by separate steps in classical schemes. Fourteen pyrethroid pesticides, including tefluthrin, tetramethrin, cyphenothrin, cyfluthrin, flucythrinate, tau-fluvalinate, deltamethrin, bioallethrin, fenpropathrin, lambda-cyhalothrin, permethrin, alpha-cypermethrin, esfenvalerate and tralomethrin were determined using the described procedure with satisfactory recoveries for most of them, at spiking levels ranging from 0.08 to 0.82 mg/kg for the different compounds. Crops subjected to the described procedure included strawberry, apple, and orange gave extracts containing a mass of co-extractives that was between 5 and 30 mg. Compared with classical schemes, the described procedure is simple, less labour intensive, allows parallel handling of several extracts and does not require the preparation and maintenance of equipment. Troublesome emulsions such as those frequently observed in separation funnel partitioning do not occur.

Selective extraction of pyrethroid pesticide residues from milk by solid-matrix dispersion.

A rapid procedure has been developed that allows a single-step, selective extraction and clean-up of pyrethroid (PYR) pesticide residues from milk dispersed on solid-matrix diatomaceous material filled into disposable cartridges and eluted by means of light petroleum saturated with acetonitrile and ethanol. The extract was cleaned up by high-performance size-exclusion chromatography. Determinations were carried out by gas chromatography with electron-capture detection. Recovery experiments were carried out on homogenized commercial milk (3.6% fat content) that was spiked with solutions of 14 PYR pesticides, viz., tefluthrin, tetramethrin, cyphenothrin, cyfluthrin, flucythrinate, fluvalinate, deltamethrin, bioallethrin, fenpropathrin, lambda-cyhalothrin, permethrin, alpha-cypermethrin, esfenvalerate and tralomethrin, at levels ranging from 0.04 to 0.41 mg/kg for the different PYR pesticides. Average recoveries were in the range 60-119% for the different PYR pesticides, with relative standard deviations from ca. 2.5 to 14.4%. Coextracted fatty material amounted to an average of ca. 5 mg/ml of milk. The sole extraction step requires about 30 min. The main advantages of the procedure are that extraction of PYR pesticides (with a minimum carry over of fat) is performed in a single step, emulsions do not occur, several samples can be run in parallel by a single operator, reusable glassware is not needed and simple operations are required.

Selective, solid-matrix dispersion extraction of organophosphate pesticide residues from milk.

A rapid procedure has been developed that allows a single-step, selective extraction and cleanup of organophosphate (OP) pesticide residues from milk dispersed on solid-matrix diatomaceous material filled into disposable cartridges by means of light petroleum saturated with acetonitrile and ethanol. Recovery experiments were carried out on homogenized commercial milk (3.6% fat content) spiked with ethanolic solutions of 24 OP pesticides, viz., ethoprophos, diazinon, dimethoate, chlorpyrifos-methyl, parathion-methyl, chlorpyrifos-ethyl, malathion, isofenphos, quinalphos, ethion, pyrazophos, azinphosethyl, heptenophos, omethoate, fonofos, pirimiphos-methyl, fenitrothion, parathion, chlorfenvinphos, phenthoate, methidathion, triazophos, phosalone, azinphos-methyl, at levels ranging for the different OP pesticides from 0.02 mg/kg to 1.11 mg/kg. Average recoveries of four replicates were in the range 72-109% for the different OP pesticides, with relative standard deviations (R.S.D.) from ca. 1 to 19%, while dimethoate and omethoate were not recovered. Coextracted fatty material amounted to an average of about 4.0 mg/ml of milk. The extraction procedure requires about 30 min. The main advantages are that extraction and cleanup are carried out in a single step, emulsions do not occur, several samples can be run in parallel by a single operator, reusable glassware is not needed and simple operations are required.

Determination of pyrethroid pesticide residues in fatty materials by solid-matrix dispersion partition, followed by mini-column size-exclusion chromatography.

The method studied uses a combination of a solid-matrix dispersion partition (SMDP) followed by high-performance size-exclusion chromatography on a minicolumn (HPmSEC) of 7.8 mm I.D. for the separation of pyrethroid (PYR) residues from fatty material. The solid-matrix dispersion extraction is carried out by absorbing a fat solution onto an Extrelut-3 cartridge (filled with a macroporous diatomaceous material) and extracting the PYR residues with acetonitrile. Up to 1 g of fatty material can be extracted with 15 ml acetonitrile. The small amount (mean +/- S.D. = 12.4 +/- 5.9 mg) of fatty material which is eluted into the acetonitrile is further removed by HPmSEC. PYR pesticide residues are collected in a 2-ml fraction between 7 and 9 ml, the column being washed up to 24 ml. The two techniques used in series allow a better removal of fat, a greater input of sample and a lower consumption of solvent compared to the sole SEC on macrocolumns, and a lower limit of determination compared to the sole SEC on minicolumns. Recoveries of 9 PYR out of the 14 investigated residues from soya oil were in the range 66-83% at spiking levels ranging 0.49-2.57 mg/kg, while for 6 PYR residues tested at spiking levels in the range 0.13-0.53 mg/kg the recoveries were in the range 80-111%. Recovery of fluvalinate and permethrin could not be calculated due to interferences from soya oil, while lambda-cyhalothrin, esfenvalerate and tralomethrin gave low recovery. The final extract contains small amount (mean +/- S.D. = 2.4 +/- 0.9 mg) of lipid residue and is not completely free from interferences.

Selective clean-up applicable to aqueous acetone extracts for the determination of carbendazim and thiabendazole in fruits and vegetables by high-performance liquid chromatography with UV detection.

Fungicide residues in vegetables (benomyl, carbendazim, thiabendazole) are analyzed through a clean-up procedure that uses a portion of the aqueous acetone extract prepared for multiresidue methodology. A portion of the aqueous acetone extract (equivalent to 5 g of vegetables) is loaded onto an Extrelut-20 cartridge (the cartridge is filled with a coarse, large-pore diatomaceous material). Then, acetone is partially removed by an upward stream of nitrogen at 2l/min for 30 min. Benzimidazolic fungicides are recovered by percolating the cartridge with 100 ml of 0.1 M phosphoric acid solution, which also serves to convert benomyl to carbendazim. The percolating acid solution is drained on-line through a strong cation-exchange (SCX) solid-phase extraction cartridge with the aid of a slight vacuum. Benzimidazolic fungicides are retained on the SCX cartridge. The phosphoric acid solution is discarded together with the washings of the SCX cartridge, i.e., water followed by methanol-water (75:25), that remove unwanted coextractives. Finally, benzimidazolic fungicides are recovered by eluting the SCX cartridge with methanol-ammonium formate buffer (75:25). The final extract is then analyzed by reversed-phase HPLC with UV detection. Recoveries from crops such as apples, lettuce, strawberries and citrus fruits are generally greater than 80% and no interferences were observed. The clean-up is simple and straightforward, requires only disposable items, water solutions and a few milliliters of solvent and a minimum number of manipulations, and does not require concentration steps or electrical equipment.

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells.

DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.

Distribution of hydrocarbons in bovine tissues.

Liver, heart, kidneys, muscle and adipose (perirenal and subcutaneous) tissues were collected from six animals for analysis of their hydrocarbon composition. Qualitative and quantitative determinations were carried out by gas chromatography and combined gas chromatography-mass spectrometry. Although differing in the proportions, a homologous series of n-alkanes ranging from n-C12 to n-C31 was found in all the samples examined. The isoprenoid hydrocarbons phytane and phytene (phy-1-ene and phyt-2-ene) were also identified.

Pesticide residue control in the years 1980-1982.

Data on pesticide residue on vegetables, fruits, cereals, and other foods of vegetable or animal origin determined by Provincial Laboratories during the years 1980-1982 are presented and compared with levels permitted by Italian legislation. An analysis of the results obtained from about 3000 samples shows that a high percentage of samples did not contain detectable pesticide residues or had residue levels below the maximum allowed by Italian law: 98% of the vegetables, 94.6% of the fruit, and 97.5% of the cereals (fumigants not considered here). On fruit, some residues exceeding the permissible levels for postharvest compounds, i.e., ethoxyquin, ethylenebisdithiocarbamate (EBDC), and benzimidazolmethylcarbamate (BMC), were found in apples and pears. Residue levels of some organochlorinated compounds slightly exceeding the legal limit were observed in some food samples of animal origin (mainly cow's milk). Comparing the present results with those of previous studies indicates that there has been a general decline in the residue levels of aldrin/dieldrin in milk and milk products.

Pesticide residue control in the years 1986-1987.

Results of analytical surveys on pesticide residue levels in foods for the years 1986-1987 are presented. Analytical data obtained from regional laboratories are tabulated in terms of various commodity groups (vegetables, fruit, cereals, processed or derived foodstuffs, other commodities, legumes, environmental samples, others) and are compared with maximum legal tolerances established by the ministerial ordinance (Italian Ministry of Health) 6.6. 1985. On a total number of 4715 determinations of classes of compounds or of single compounds made on 1601 commodities samples, only N 53 (1.1%) violative results appeared. No residue was found at levels exceeding regulatory limits or found in commodities where a tolerance had not been established for a specific pesticide/commodity combination.

Pesticide residue control in the years 1988-1989 in Italy.

The paper reports the monitoring for pesticide residue contamination of foodstuffs carried out in the years 1988-1989 by the Italian peripheral laboratories officially commissioned for the task. The tables show the analytical data for each class of substrate versus the limits in force at the time. 2506 food samples and 555 samples from the environment were analysed and a total of 16,259 determinations made. The residues were absent in 90% of the cases; the percentage of irregularities was 1.52%. These results confirm the data obtained in previous years, which is particularly significant considering the much larger number of analytical determinations performed this time. It has been deemed convenient to survey specific types of foodstuffs (for example, cereals and animal products) by examining a larger number of samples.

Pesticide residue control in the years 1983-1985.

Levels of organochlorinated, organophosphate, dithiocarbamic, benzimidazolic, and phthalimidic compounds, as well as some other pesticide residues obtained from Regional Laboratories in the years 1983-1985, were assembled and presented as a part of a study aimed at evaluating food contamination. Approximately 2000 samples of vegetables, fruits, cereals, and food of animal origin were analyzed by local laboratories during the years 1983, 1984, and 1985. Samples were randomly obtained from the market, at production levels, or sometimes at custom sites. Approximately 10,000 determinations of single compounds or classes of compounds were carried out. The analytical results are presented and compared with the Italian maximum residue limits. In the majority of the samples analyzed, either there were no residues or the residues were below the permitted level for the specific pesticide in each commodity. High levels of residues were present in a few cases, mainly with respect to some fungicides in fruits and vegetables and chlorinated compounds in some animal products.

The origin of DNA single strand breaks induced by ethylating agents in mammalian cells.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.

Evaluation of p,p'-DDE, p,p'-DDT and polychlorobiphenyls (PCBs) levels in samples of human milk from Rome, Florence and the surrounding areas.

Sixty-five samples of human milk obtained from individual donors living in Rome and surrounding areas between 1982 and 1984, and 28 samples from Florence and surrounding areas obtained during 1985 were analyzed for residues of p,p'-DDE, p,p'-DDT and PCBs. Levels of p,p'-DDE were between 5 and 126 ppb (micrograms/kg of milk), with an average value of 45 ppb (median 34). Levels of p,p'-DDT ranged from 1 to 79 ppb, with an average value of 10 ppb (median 7). PCBs were found at levels ranging from 7 to 304 ppb, with an average value of 74 ppb (median 66). As to the p,p'-DDT, when compared to the data previously obtained in Italy during 1975-77, the present findings show a decrease of the average value and a lower incidence of samples with higher values. These effects are less pronounced for the p,p'-DDE. No relevant variation was observed in the levels of PCBs in comparison with the data obtained in Italy during 1981-82.

[Estimate of the dose of chlorinated pesticides consumed daily by the Italian population (1978-1984)]. / Una stima della dose di pesticidi clorurati assunta quotidianamente dalla popolazione italiana (1978-84).

This work reports an estimate of the doses of chlorinated pesticides taken with a total diet by the Italian population in the period 1978-84. Previous researches, carried out in 1971-72, had regarded both uncooked and cooked diets, ready for the consumption. It was found that the amount of the considered pesticides taken in total (HCB, gamma-BHC and total-BHC, Aldrin/Dieldrin, Heptachlor/Heptachlor-epoxide and DDT total-equivalent) calculated as a sum of the percentage of each acceptable daily intake (A.D.I.), at that time recommended was near the limit of acceptability (98.5% for cooked diets and 107.0% for uncooked diets). In addition it was found that nearly the 40% of the dose assumed in all came from milk and its derivates (butter, cheese). Then considering the contribution of milk and derivates to the amount of pollutants assumed with a total diet, a new evaluation has been done, with regard to the period 1978-84, i.e. after the prohibition of the use of these pesticides in Italy and in other industrialized countries. Then the data have been processed, with regard to 211 samples of foods (90 of milk, 45 of cheese, 76 butter) coming from determinations carried out by the Multizonal Laboratory of Hygiene and Prevention of Ferrara, Milano, Udine, Bolzano, Trento, Pescara and from the Institute of Hygiene of Rome University "La Sapienza" in the period mentioned. Furthermore, alimentary consumptions of milk, butter and cheese in 1978-84 have been evaluated, and amounts of chlorinated pesticides taken with these foods have been calculated. Finally the dose taken with these foods has been extrapolated to a total diet. It has come out that the doses (estimated) taken with the total diet have notably decreased between 1971-72 and 1978-84. An anomaly is yet represented by HCB: while the quantity taken with food has decreased from 1971-72 to 1978-84 (from 3.7 to 1.3 micrograms/man/day), the concentrations in human adipose tissues have increased (from 1.02 to 1.99 mg/Kg). It can reasonably mean that assumption of HCB is not prevalently alimentary, but that it is due to other factors of environmental contamination. The data of the doses of chlorinated pesticides taken have also been compared with the respective ADI indicated in 1971-72 and in 1978-84.