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The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA.

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratio's by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).

Animals↗

Inhibition of acetyl-coenzyme A dependent activation of N-hydroxyarylamines by phenolic compounds, pentachlorophenol and 1-nitro-2-naphthol.

Pentachlorophenol (PCP) and 1-nitro-2-naphthol were found to be potent inhibitors of enzymatic acetyl-CoA dependent activation, which is suggested as proceeding through direct O-acetylation, of N-hydroxyarylamines to tRNA binding by liver cytosolic enzymes from hamsters and rats. IC50 values of PCP for the activation of 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) and N-hydroxy-2-aminofluorene (N-OH-AF) were 20, 25 and 17 microM, respectively, in hamster cytosol system. Similar inhibition was observed with rat liver cytosol (IC50 values of PCP and 1-nitro-2-naphthol were 13 and 12 microM, respectively, for the binding of N-OH-Glu-P-1). PCP is known as an inhibitor of sulfotransferase; however, another potent inhibitor of sulfotransferase, 2,6-dichloro-4-nitrophenol, did not inhibit the acetyl-CoA dependent binding. Antibiotic thiolactomycin, which inhibits bacterial O-acetyltransferase, did not affect the activation by hamster and rat cytosol, indicating the difference in property between bacterial and mammalian enzymes. The kinetic data obtained with hamster cytosol suggested the competitive inhibition of PCP with substrate, N-OH-Glu-P-1, and non-competitive inhibition with acetyl-CoA. In addition to the O-acetylation, PCP and 1-nitro-2-naphthol also inhibited N-acetylation of arylamines and N,O-acetyltransfer reaction of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) by hamster cytosol. IC50 values for these two types of acetyltransfer reactions, however, were slightly higher than those observed for acetyl-CoA dependent activations of N-hydroxyarylamines.

Acetyl Coenzyme A↗

Pentachlorophenol-induced change of zeta-potential and gel-to-fluid transition temperature in model lecithin membranes.

We have determined zeta-potentials for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) membranes by measuring the electrophoretic mobility of multilayered vesicles and the temperatures of the gel-to-ripple-to-fluid phase transitions of sonicated vesicles by a photometric method. Some conclusions are: (1) The zeta-potentials of DMPC and DPPC vesicles become negative due to adsorption of ionized pentachlorophenol (PCP), (2) their magnitude changes, step-like, on gel-to-fluid transition and (3) the temperature of the step-like change in zeta-potential decreases with an increase in PCP concentration. (4) PCP exhibits a large effect on membrane structure: It induces an isothermal phase change from the ordered to disordered state, which is enhanced by monovalent salt in the aqueous phase. (5) Both ionized and unionized PCP decrease the melting phase transition temperature and abolish the pretransition, (6) the unionized species increases the melting transition width and (7) the ionized species is more potent in abolishing the pretransition. (8) The shorter chain lipid (DMPC) is more sensitive to the presence of PCP; the maximum decrease in delta Tt is 13 K (DMPC) and 7 K (DPPC) in the presence of ionized PCP. We have shown experimentally, by comparing the delta Tt from photometric studies with the density of adsorbed PCP derived from zeta-potential isotherms, that (9) the shift of the melting phase transition temperature increases linearly with the density of adsorbed PCP. (10) In contrast to membranes made of negatively charged lipids, the transition temperature of DMPC and DPPC membranes in the presence of PCP further decreases in the presence of monovalent salt. The salt effect is due to screening of the membrane surface leading to enhanced adsorption of ionized PCP and a depression in transition temperature. (11) It is shown that both the adsorption and the changes of gel-to-fluid phase transition temperature can be described in terms of the Langmuir-Stern-Grahame model and (12) proposed that future studies of membrane toxicity of PCP should be focused on its pH dependence.

1,2-Dipalmitoylphosphatidylcholine↗

Role of active oxygen species in DNA damage by pentachlorophenol metabolites.

Pentachlorophenol (PCP) has been shown to be carcinogenic for mice, although it does not seem to be mutagenic in bacterial test systems. In this study, the mechanism of DNA damage by PCP metabolites in the presence of metals was investigated with a DNA sequencing technique using 32P-labeled DNA fragments and with an electrochemical detector coupled to an HPLC. The metabolite tetrachlorohydroquinone (TCHQ) caused DNA damage in the presence of Cu(II) but not in the presence of either Mn(II) or Fe(III). TCHQ plus Cu(II) frequently induced piperidine-labile sites at thymine residues and guanine residues. The most preferred sites were the thymine residues of the 5'-GTC-3' sequence. TCHQ increased 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Typical OH scavengers showed no inhibitory effects on TCHQ- plus Cu(II)-induced DNA damage. Bathocuproine and catalase inhibited DNA damage, suggesting that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. Tetrachloro-p-benzoquinone (TCBQ) alone did not induce DNA damage in the presence of Cu(II), but addition of NADH induced DNA cleavage even in the absence of NADH-FMN oxidoreductase. UV-visible and ESR spectroscopies have demonstrated that TCHQ is rapidly autoxidized into semiquinone even in the absence of metal ions, indicating that the semiquinone radical itself is not the main active species inducing DNA damage. These results suggest that the semiquinone radical produced by the autoxidation of TCHQ and/or the reduction of TCBQ by NADH reacts with dioxygen to form superoxide and subsequently H2O2, which is activated by transition metals to cause DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗

Co-recombinogenic and anti-mutagenic effects of diethylhexylphthalate, inactiveness of pentachlorophenol in the spot test with mice.

In in vivo experiments using the spot test with mice, diethylhexylphthalate (DEHP) was co-recombinogenic and anti-mutagenic. In two independent experiments, DEHP was able to increase in combination with ethylnitrosourea (ENU) the frequency of animals with spots of genetic relevance from about 12% (ENU alone) to about 15% (ENU+DEHP). This enhancement can be exclusively attributed to an enhancement of recombination. In historical as well as in current experiments, with astonishing accuracy, about 8% of all spots induced with ENU alone are black, near-white or twin spots, i.e., presumed products of reciprocal recombination. Under the influence of DEHP, about 20% of all colored spots were black, near-white or twin spots. These co-recombinogenic effects are very strong, considering that the low frequency of twin spots with 0.3% (historical ENU control) or 0.6% (current ENU control) after ENU treatment alone has been enhanced up to a frequency of 3%. While ENU alone induces about 14% light brown colored spots, depending exclusively on gene mutations, under the influence of DEHP the frequency was only 7%, i.e., DEHP was anti-mutagenic. Pentachlorophenol was ineffective in enhancing or reducing recombination or mutations. There was no difference between ENU and ENU+PCP treatment. Since a number of "non-genotoxic' carcinogens with tumor-promoting activities like D-limonene, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) have shown similar effects in the spot test, it is suggested that DEHP may have tumor-promoting activity in carcinogenicity tests.

Animals↗

An investigation of the sensitivity of the ouabain-insensitive sodium efflux in single barnacle muscle fibers to pentachlorophenol.

The aim of the present work was to explore the possibility that pentachlorophenol (PCP) influences the behavior of the resting Na efflux in single muscle fibers from the barnacle, Balanus nubilus. It is shown here that PCP causes a transitory rise in the Na efflux in both unpoisoned and ouabain-poisoned fibers and that the response is dose-dependent, the minimal effective concentration in ouabain treated fibers being less than 10(-6) M. The efficacy of PCP is significantly greater than that of 2,3,4-trichlorophenol. 2,3-Dichlorophenol is ineffective. This is also the case with phenol. The magnitude of the response to PCP is a function of external pH. Lowering pHe increases the response. The response has an absolute requirement for external Ca2+ and is a sigmoidal function of external Ca2+ concentration. Since treatment of these fibers with PCP in high concentration leads to prompt contraction, experiments were designed to determine whether the observed rise in ouabain-insensitive Na efflux is due to a fall in myoplasmic pCa and whether trigger Ca2+ originates from the bathing medium. The results obtained show that prior injection of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA) leads to a drastic reduction in the response to PCP. They also show that prior external application of verapamil or devapamil stops the response to PCP from occurring. Both Cd2+ and Co2+ are also effective but only temporarily. Last, the effects of ryanodine and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were tested, since the former is known to block the sarcoplasmic reticulum Ca2+ release channel, and the latter to impair the action of agents known to release Ca2+ from internal depots. Both ryanodine and TMB-8 are found to reduce the response to PCP. Taken together, these observations support the hypothesis that PCP stimulates the ouabain-insensitive Na efflux by increasing the internal free Ca2+ and that the increase in internal Ca2+ is due to the entry of trigger Ca2+ from the outside via Ca2+ channels, as well as release of Ca2+ by the sarcoplasmic reticulum via its channel. They also indicate that the efficacy of PCP depends on the 5 Cl atoms present in its aromatic ring and pHe.

Animals↗

Comparison of dibenzodioxin levels in blood and milk in agricultural workers and others following pentachlorophenol exposure in China.

Substantial amounts of sodium pentachlorophenol (Na-PCP) salts have been sprayed in certain areas in central China since the 1960s for control of snail-borne schistosomiasis. A sample of Na-PCP used in the area of concern was analyzed and showed 2,3,7,8-substituted dibenzodioxin (PCDD) and dibenzofuran (PCDF) congeners as well as some non-2,3,7,8-substituted congeners. Dibenzodioxin analysis of two pooled blood samples from 100 persons living in sprayed areas and a pooled sample from 26 individuals who had direct contact with the Na-PCP showed elevations of PCDD/F congeners found in the Na-PCP. Total PCDD/Fs ranged from 631 to 1252 parts per trillion (ppt) lipid in the blood of those exposed to Na-PCP, while two general population age-matched pooled blood samples from 50 persons each had total PCDD/Fs of 147 and 178 ppt. Toxic equivalents calculated with International Toxic Equivalency Factors (I-TEqs) ranged from 9 to 16.3 ppt in the blood samples from exposed persons while the general population blood I-TEqs were 4.8 and 6.4 ppt. A pooled breast milk sample from 50 women living in the sprayed areas was compared with a pooled sample from 50 women living in unsprayed areas. Total PCDD/Fs was low in both exposed and general population samples (134 and 42 ppt lipid, respectively), however, the women living in sprayed areas had considerably higher PCDDs, 129 ppt versus 34 ppt. The mothers exposed to Na-PCP had a I-TEq of 5.4, which was about double that of the mothers from the general population, 2.6 ppt, lipid. Although human PCDD and PCDF tissue levels in China are low compared with those in more industrialized countries, the higher levels in exposed persons are cause for concern.

Adolescent↗

Pentachlorophenol accumulation in the freshwater mussels Anodonta anatina and Pseudanodonta complanata, and some physiological consequences of laboratory maintenance.

Freshwater mussels Anodanta anatina and Pseudanodonta complanata were exposed to (14C)-pentachlorophenol. The wet weight based bioconcentration factor (BCF = activity in animal per activity in water) at steady state varied from 80 to 120 for A. anatina and from 61 to 85 for P. complanata. The species did not differ significantly in their wet weight or lipid based BCFs but dry weight based values were significantly higher (40-50%) for A. anatina. The soft tissue dry weight and dry weight based condition index of A. anatina (Cl4 = soft tissue dry weight per shell length) differed significantly between natural mussel populations. In animals kept from 4 to 8 months in laboratory conditions, the soft tissue dry weight and glycogen content decreased more rapidly when mussels were maintained at 15 than at 5 degrees C. However, glycogen content in the digestive gland or adductor muscle did not differ in mussels maintained in the laboratory (5 degrees C) when compared to the natural population. The adductor muscle protein content differed between laboratory maintained animals and the natural population in Lake Höytiänen but there was no difference in the soft tissue lipid content. Trace metal concentrations and calcium in the soft tissue were in general higher in laboratory maintained mussels. In addition, laboratory maintenance affected the reproductive cycle of A. anatina.

Animals↗

Determination of pentachlorophenol in commercially prepared lyophilized human urine control samples.

Commercially available reference materials consisting of lyophilized human urine are routinely utilized by clinical chemists to monitor laboratory performance for a variety of analyses. In this study commercially prepared lyophilized human urine control samples were reconstituted and analyzed for pentachlorophenol. Based on replicate analyses of two different reference materials, the levels of PCP in each were found to be representative of the levels typically present in urine samples collected from the general population of the province of Saskatchewan.

Humans↗

Uptake and accumulation of pentachlorophenol and sodium pentachlorophenate by earthworms from water and soil.

Toxicity values were obtained for pentachlorophenol (PCP) and other compounds in the standard OECD and EEC earthworm test. To help explain the uptake and bioaccumulation mechanisms of PCP which affect toxicity, experiments have been conducted using the earthworm Allolobophora caliginosa. The experiments showed that radiolabelled PCP and its sodium salt, PCP-Na, were taken up rapidly by the earthworms during 24 h from aqueous solution (1 and 10 micrograms ml-1) in a limited volume (5 ml) to give a bioconcentration factor (BCF) of 2.5, and that the solution concentration had no apparent influence on uptake. Exposure of worms over a period of 14 days in a large volume of soil under laboratory conditions increased the BCF, which was approximately 8 and 13 in the low (2.2 mg g-1 soil) and high (11.2 mg g-1 soil) soil concentration respectively. Longer exposures of the same worm species for 131 days in another soil type, in lysimeters under outdoor conditions, did not increase the BCF beyond that reached after 14 days exposure in the artificial soil. There were differences between earthworm species in their bioconcentration ability: Lumbricus terrestris accumulated 3 times more radiocarbon than A. caliginosa. Irrespective of the concentrations in solution, the binding of free phenol (PCP) in worm tissues was greater than for its sodium salt (PCP-Na), but there were no apparent differences in the amount of these substances eliminated, which was rather low. Metabolic studies have shown that PCP is metabolised more rapidly than PCP-Na in the worms body, resulting in the formation of metabolites which are polar in nature and also probably of conjugates. The results are discussed in relation to the mechanisms which may be responsible for the detoxification of PCP in earthworms.

Animals↗

Relationship between the biodegradative capability of soil micromycetes for pentachlorophenol and for pentachloronitrobenzene.

A collection of 1056 strains in our laboratory were incubated with various xenobiotics among which were two potent fungicides: pentachlorophenol (PCP) and pentachloronitrobenzene (PCNB). The production of extracellular phenoloxidases were examined, using a series of ten different reagents. On the whole, PCNB is less accessible to fungal degradation than PCP. Although no correlation was found between the biodegradative capability of individual fungal strains for PCP or for PCNB, when taxonomic groups were considered as a whole, the same activity profiles were found. Zygomycetes were the most efficient; yeasts the least efficient towards both substrates. A more detailed study of the metabolism of both substrates on selected strains is in progress.

Biodegradation, Environmental↗

Analysis for nonextractable (bound) residues of pentachlorophenol in plant cells using a cell wall fractionation procedure.

When plant cell cultures or aseptically grown wheat plants were treated with [14C]-pentachlorophenol (PCP) a major part of the label was found in a nonextractable or "bound" residue fraction. Soluble polar conjugates participated in the formation of these residues which were mainly located in the plant cell walls. By a sequential fractionation procedure using enzymatic and chemical methods, 90 to 95% of the bound radioactivity could be attributed to individual cell wall components. The 14C label from PCP was found mainly in hemicellulose, lignin, and protein fractions. Associations of hemicellulose with PCP derivatives with molecular weights up to 500,000 were purified to constant specific radioactivity. Hydrolysis of this fraction released 32% PCP and other unidentified products.

Adsorption↗

Effects of long-term exposure to pentachlorophenol on the free amino acid pool and energy reserves of the freshwater amphipod Gammarus pseudolimnaeus Bousfield (Crustacea, Amphipoda).

The freshwater amphipod Gammarus pseudolimnaeus was exposed for 45 days to pentachlorophenol (PCP). The total concentration of free amino acids (FAA) decreased significantly after 5 days exposure to 0.77 or 1.25 mg PCP/liter. Exposure to PCP had no effect on the relative concentrations of individual amino acids exposed to 0.77, 1.06, or 1.25 mg PCP/liter. Whole body concentrations of glycogen, protein, and caloric content were significantly decreased after 15 days and lipid content after 30 days exposure to 0.77 mg PCP/liter. Since energy reserves and caloric content were not measured on the 5th day of PCP exposure, the relative sensitivity of FAA as a biochemical indicator of toxicant-induced stress cannot be compared directly. However, since reductions in growth and/or energy reserves can significantly influence the fecundity of amphipods, alterations in the FAA pool are indicative of future adverse effects on the organism. The use of alterations in the concentration of FAA is discussed with respect to its application as a biochemical indicator of toxicant exposure in freshwater invertebrates.

Amino Acids↗

Effects of pH on the acute toxicity and uptake of [14C]pentachlorophenol in the midge, Chironomus riparius.

The acute toxicity of pentachlorophenol (PCP) was determined at pH levels 4, 6, 9 to the midge, Chironomus riparius, with the findings that PCP is of greatest toxicity at pH 4 and of least toxicity at pH 9. This differential toxicity is attributable to variations in uptake levels at the respective pH levels. At pH 4, PCP is fully protonated and therefore highly lipophilic. The amount of [14C]PCP present in the midges at 24 hr is thus highest at pH 4. Conversely, at pH 9, the compound is completely ionized. The reduction in lipophilicity at pH 9 decreases the ability of the compound to penetrate into the midge, thereby decreasing the observed toxicity of the compound.

Animals↗

The effect of pentachlorophenol and its metabolite tetrachlorohydroquinone on RNA, protein, and ribosome synthesis in Saccharomyces cells.

The effect of pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCH) were tested on growth, RNA, protein and ribosome syntheses, and ribosome content in yeast cells. Cells exposed to increasing concentrations of PCP show increasing inhibition to RNA and ribosome synthesis, and to cell growth. TCH causes a delay of the growth of the cell culture (prolongation of the lag phase) but does not cause inhibition. After treatment with TCH the maximum of the RNA synthesis was retarded, but subsequently reached nearly the same level as the untreated control cells. On ribosome synthesis and ribosome content, treatment with increasing concentrations of PCP, as well as of TCH, leads to a substantial decrease in ribosomal synthesis and, finally, total inhibition. Parallel to this, the content of free and membrane-bound ribosomes is diminished. PCP exhibits a stronger effect than TCH. The protein synthesis is only slightly reduced after treatment with PCP or TCH (with concentrations up to 20 micrograms/ml).

Chlorophenols↗

Biodegradation potential of some micromycetes for pentachlorophenol.

A first screening was performed upon 100 strains of micromycetes cultivated on solid media (malt extract medium and mineral medium) with pentachlorophenol (PCP) (0.5 g/liter). Under these conditions, 50 strains gave a light blurring around the inoculation spot, indicating some PCP degradation. Later, 50 selected strains were cultivated in liquid synthetic medium with PCP (1 g/liter). After 6 days of cultivation, photodegradation occurred for 25%. On the whole, the consumption of PCP was 25% for Zygomycetes, 3% for yeasts, and 10-15% for Deuteromycetes, except 7% for Tuberculariales. It was shown that glucose repressed the PCP consumption. Among degrading fungi, some could grow with PCP when cultures were initiated with spores, others could not. A more detailed study was done with Phoma glomerata cultivated in liquid synthetic medium (PCP 100 mg/liter) in darkness or with light. Photodegradation increased up to 25% but abiotic degradation occurred also in darkness (8%). Consumption of PCP by Ph. glomerata was 27% after 2 days with light and was lower in darkness (19%).

Biodegradation, Environmental↗

Pentachlorophenol.

Pentachlorophenol (PCP) is a substance whose widespread use, mainly in wood protection and pulp and paper mills, has led to a substantial environmental contamination. This in turn accounts for a significant exposure of the general human population, with rather high exposure levels being attained in occupational settings. Investigations on the genotoxic activity of PCP have given rise to divergent results which would seem to make an evaluation difficult. By grouping them into 3 categories a somewhat clearer picture, allowing finally an (admittedly tentative) assessment, can be obtained. PCP does seem to be at most a weak inducer of DNA damage: it produces neither DNA-strand breaks nor clear differential toxicity to bacteria in rec-assays in the absence of metabolic activation. Also in SCE induction no increase can be observed in vivo, while PCP is found marginally active in a single in vitro experiment. Metabolic activation, however, leads to prophage induction and to DNA strand breaks in human lymphocytes, presumably through the formation of oxygen radicals. A possible further exception in this area might be the positive results in the yeast recombination tests, although their inadequate reporting makes a full evaluation difficult. PCP does not seem to induce gene (point) mutations, as most bacterial assays, the Drosophila sex-linked recessive lethal test and in vitro assays with mammalian cells did not demonstrate any effects. Marginally positive results were obtained in the mammalian spot test in vivo and in one bacterial test; the positive result in the yeast assay for cycloheximide resistance is fraught somewhat with its questionable genetic basis. PCP does, however, induce chromosomal aberrations in mammalian cells in vitro and in lymphocytes of exposed persons in vivo. Those in vivo results that were unable to provide evidence of chromosomal damage are hampered either by methodological inadequacies or by too low exposure levels. The (rodent) metabolite tetrachlorohydroquinone might be a real genotoxic agent, capable of binding to DNA and producing DNA strand breaks; this activity is probably due to semiquinone radical formation and partly mediated through active oxygen species. Since this compound has not been tested in the common bacterial and mammalian mutagenicity assays, the few ancillary results on this substance cannot be used in a meaningful human risk assessment of PCP. Furthermore, this metabolite has only been produced by human liver microsomes in vitro, but has not been detected in exposed humans in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Chromosome changes in lymphocytes after occupational exposure to pentachlorophenol (PCP).

Chromosome analyses were carried out on peripheral lymphocytes from 22 male workers employed at a pentachlorophenol (PCP) producing factory. As compared with a group of 22 matched controls a small, but significant, increase in the frequency of dicentrics and acentrics was observed. There was no significant increase of sister-chromatid exchange (SCEs) in smoking PCP workers, as compared with smoking controls. Within the control group, smokers had a higher incidence of SCEs than non-smokers.

Air Pollutants↗