Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).     

Measurement of spontaneous and X-irradiation-induced 6-thioguanine-resistant human blood lymphocytes using a T-cell cloning technique

Lymphocytes separated from human peripheral blood were cultured in vitro, in the presence of 6-thioguanine (TG), to select and clone rare TG-resistant (TGr) cells present in the circulation in vivo. The incidence of such TGr cells ranged from 0.83 X 10(-5) to 2.53 X 10(-5) (mean 1.48 X 10(-5) ) in healthy individuals aged between 19 and 79 years; did not differ between males and females; but increased significantly with age at a rate of 2.4 cells/10(7) lymphocytes/year. Exposure of lymphocytes (G0) in vitro to X-ray doses of upto 200 rad resulted in a dose-dependent increase in TGr cell frequencies. The rates of increase were approximately in proportion to the square of the dose and these rates were closely similar to those obtained in cultured skin fibroblasts and suggest that the bulk of these mutations are a consequence of chromosome structural aberrations. The cloned TGr cells are considered to be HPRT- mutants and the mutation frequencies in lymphocytes determined using this cloning technique were compared with the variant frequencies obtained in earlier experiments utilising an autoradiographic technique to detect azaguanine-resistant (AGr) variant cells. Mutation frequencies with the cloning technique were 10-20-fold lower than variant frequencies with the autoradiographic method.     

Enumeration of 6-thioguanine-resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo.

An autoradiographic method to enumerate variant 6-thiogunanine-resistant (TGr) peripheral blood lymphocytes (PBLs) that occur in vivo in man is described. Variant cells are detected in PBL cultures stimulated to tritiated thymidine (3HTdr) incorporation in vitro with phytohemagglutinin (PHA) in the presence of TG. Cells with the naturally-occurring Lesch--Nyhan (LN) mutation served as prototype-variant cells. PBLs from a LN hemizygous male were found to be resistant to TG inhibition of PHA-stimulated 3HTdr in corporation in vitro while a LN heterozygous female was found to be a mosaic with 2/1000 PBLs resistant to 2 X 10(-4) M TG. Experiments with artificial mixtures of LN and normal PBLs showed that the LN cells were virtually all detectable even when present in low frequency (10(-5)). TGr PBLs were found in healthy non-LN individuals at median frequencies of 1.0 X 10(-4) and 1.1 X 10(-4) when determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG respectively. Their frequencies were not age-related. TGr PBL-variant frequencies (Vf's) were determined in 47 cancer patients who were being treated with cytotoxic agents that are known to be mutagens. The median TGr PBL Vf determined at 2 X 10(-3) M TG in cancer patients was 2.2 X 10(-4) while, when determined at 2 X 10(-4) M TG, it was 8.5 X 10(-4). The distribution of Vf's for the treated cancer-patient group differed from that for the normal control group in that more than half of the treated cancer patients had TGr PBL Vf's greater than the highest seen for controls. Unlike those of the normal controls, the TGr PBL Vf's of treated cancer patients differed if determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG, a behavior that suggested partial resistance and mimicked that seen with LN TGr PBLs. PBLs resistant to 2,6-diaminopurine (DAPr) were not found in two individuals, although the TGr PBL Vf was elevated in one. TGr PBL Vf's were greatly elevated under conditions of in vivo selection in patients receiving purine-analogue immunosuppressive therapy. The TGr PBL enumerative assay system is presented as one of potential value to detect somatic cell mutations occurring in vivo in man.     

A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

A clonogenic assay to quantify thioguanine (TG)-resistant (TGr) spleen lymphocytes in the mouse has been developed to support studies of in vivo mutation affecting the hypoxanthine phosphoribosyltransferase (hprt) locus. Lymphocytes are cultured in 96-well microtiter plates for 9 days with proliferation initiated by the mitogen concanavalin A and supported thereafter by conditioned medium containing interleukin-2. Lymphocytes are plated at high densities (4-8 X 10(5)/well) with TG and irradiated L5178Y lymphoma cells (10(4)/well) to detect the presence of TGr cells. To determine the cloning efficiency without TG lymphocytes are plated at a low density (10/well) with irradiated L5178Y cells and irradiated lymphocytes (4-8 X 10(5)/well). Proliferation of cells is detected by [3H]thymidine incorporation and scintillation spectrometry. Spontaneous frequencies of TGr clones are independent of TG dose from 0.2 to 10 micrograms/ml and independent of cell density over the range cited. The TGr clones tested have less than 10% hypoxanthine incorporation in vivo relative to unselected clones and have stable phenotypes in the absence of selection. The spontaneous frequency of TGr cells ranged from 1 to 3 X 10(-6). In vivo treatment of mice intraperitoneally with ethylnitrosourea 15 days prior to in vitro culture resulted in a linear dose-related increase of TGr cells, with 70.2 mg/kg inducing a frequency of TGr cells of 2 X 10(-5).     

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.     

6-Thioguanine resistant T-lymphocyte determination as a possible indicator of human ionizing radiation exposure

We used the autoradiographic assay to assess human in vivo somatic cell gene mutation at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in T-lymphocytes. Cells able to incorporate tritiated thymidine in vitro in 6-thioguanine containing short-term cultures were enumerated in order to determine 6-thioguanine resistant (TGr) variant frequencies in cryopreserved lymphocytes from control individuals and 3 persons suspected of 60Co exposure from an accident in Cd. Juárez, México. The data indicate that the lymphocyte TGr variant frequency assay may be potentially usefull for human population monitoring following accidental exposures to ionizing radiation.     

In vivo mutant T cell frequency in atomic bomb survivors carrying outlying values of chromosome aberration frequencies

Frequencies of HPRT- mutant T cells were determined by means of a direct clonal assay in atomic bomb survivors who showed outlying values of chromosome aberration frequencies. The studied survivors consisted of 2 groups: those whose aberration frequency was near the higher end of the distribution (high-aberration group) and those whose aberration frequency was near the lower end of the distribution (low-aberration group). The mean radiation doses (T65D) of the high-aberration group (13 people) and low-aberration group (17 people) were 248 and 273 rad, respectively. The mean mutant frequency (Mf) of the high-aberration group was 6.7 X 10(-6), which was significantly higher than that of the low-aberration group (3.7 X 10(-6)) or that of 17 controls (3.4 X 10(-6)). When all the samples were combined, the correlation between Mf and radiation dose was not significant using either dose estimation system, T65D or DS86. However, the correlation coefficient was higher when DS86 doses were used. Mf correlated significantly with increasing aberration frequencies. The tendency that Mf correlates better with chromosome aberration frequency than with estimated radiation dose was stronger in this study than in a previous study where the samples were selected randomly.     

Somatic gene mutations in vivo as indicated by the 6-thioguanine-resistant T-lymphocytes in human blood

The clonal and the autoradiographic assays for 6-thioguanine-resistant (TG^r) T-lymphocytes (T-Lys) in human blood are reviewed. Studies of TG^r colonies recovered from clonal assays show that the mutant T-Lys (i) are either helper (T4) or suppressor (T8) cells, (ii) poses stable TG^r phenotype, (iii) are deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT), and (iv) have structural alterations in the hprt gene. TG^r T-Ly mutant frequencies (Mfs) determined by clonal assays are of the order of 10⁻⁶−10⁻⁵ for normal adults. Autoradiographically determined variant frequencies (Vfs) are also in this range for normal adults when lymphocytes are cryopreserved before study to remove ‘phenocopies’. Cancer exposed to potentially mutagenic treatments have elevated TG^r T-Ly Vfs. Comparative clonal and autoradiographic assays of the same blood samples give generally similar results when allowances are made for potential sources of error in each assay. The TG^r T-Ly system is presented for human specific-locus mutagenicity monitoring.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Autoradiographic detection of mutation to exotoxin-A resistance in mouse fibroblasts treated with ethyl methanesulfonate, X-rays and ultraviolet light

P. aeruginosa exotoxin-A (PE) blocks protein synthesis in mammalian cells by inactivating elongation factor 2 (EF-2). Toxin-resistant mutant cells can be detected autoradiographically, in cultures grown on microscope coverslips in the presence of PE, and then exposed to [3H]leucine. The frequency of PE-resistant cells detected by the autoradiographic assay in non-mutagenized cells of the established mouse cell line LTKA is 9.7 +/- 0.6 X 10(-5). Upon treatment with ethyl methanesulfonate (EMS), X-rays or ultraviolet (UV) light it increases in a dose-dependent fashion. The mutational nature of the resistance detected by the assay is indicated by its clonal inheritance, and by the dose-dependent increase in the frequency of resistant cells after mutagenesis. On the basis of the high frequency of PE-resistant cells detected by the autoradiographic assay, and their cross-resistance to diphtheria toxin (DT), we suggest that the PE-resistant mutants detected by the autoradiographic assay are of class II, i.e., they are altered in the structural gene for EF-2. The autoradiographic assay for PE resistance is similar to that for DT resistance, but is applicable also to mouse cells, which are naturally resistant to DT. Being independent of colony formation, the autoradiographic assay for PE resistance can be used with non-dividing cells, either in vitro or in vivo.     

6-Thioguanine-resistant T-lymphocyte autoradiographic assay. Determination of variation frequencies in individuals suspected of radiation exposure

We used the autoradiographic assay to assess human in vivo somatic cell gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in T-lymphocytes. Cells able to incorporate tritiated thymidine in vitro in the presence of 6-thioguanine were enumerated in order to determine 6-thioguanine-resistance (TGr) variant frequencies in cryopreserved lymphocytes from 17 normal control individuals, from 3 persons suspected to have been exposed to 60Co in an accident in Cd. Juárez (Mexico), studied 24 months after the accident, and from 4 individuals who were in Kiev during the radiation accident in Chernobyl (U.S.S.R.); 2 of them were studied 1 month after the accident, and again 1 year after the first sampling, the other 2 were studied 13 months after the accident. The data obtained indicate that this assay may be useful in any laboratory of cytogenetics for human population monitoring and that its use following accidental exposure to ionizing radiation should be further evaluated.     

Mutation carrier testing in Lesch-Nyhan syndrome families: HPRT mutant frequency and mutation analysis with peripheral blood T lymphocytes

Mutations in the X chromosome hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene are responsible for Lesch-Nyhan syndrome and related diseases in humans. Because the gene is on the X chromosome, males are affected and females in the families are at risk of being carriers of the mutation. Because there are so many different mutations that can cause the disease (218 different mutations in 271 families), genetic testing for carrier status of females requires detailed molecular analysis of the familial mutation. This analysis can be complicated by the unavailability of an affected male for study. In addition, when the mutation is a deletion (34 reported instances), molecular analysis in females is difficult because of the two X chromosomes. We have applied a peripheral blood T lymphocyte cloning assay that uses resistance to the purine analogue 6-thioguanine (TG) to measure the frequency of cells in females expressing a mutant HPRT allele to determine mutation carrier status in 123 females in 61 families. In families in which the HPRT mutation was determined and could be easily analyzed in samples from females, we found a mean (+/- SD) mutant frequency of 9.7 (+/- 8.7) x 10(-6) in noncarrier females and 2.9 (+/- 3.0) x 10(-2) in carrier females. The frequency in carrier females is less than the 0.5 expected for nonrandom X inactivation because of in vivo selection against HPRT mutation-expressing T lymphocytes or stem cells during prenatal development. The use of this cloning assay allows determination of the carrier status of females even when the HPRT mutation is not yet known or is difficult to determine in DNA samples from females. This approach provides a rapid assay that yields information on carrier status within 10 days of sample receipt.     

Cloning of in vivo-derived thioguanine-resistant human B cells

In vivo-derived thioguanine-resistant (TGr) B cells have been cloned from peripheral blood mononuclear cells (PBMC) of 4 healthy adults. This was done by using Epstein-Barr (EB) virus transformation of B cells enriched from a large number of PBMC obtained with a blood cell separator. The cloned TGr B cells lacked hypoxanthine guanine phosphoribosyltransferase (HPRT) enzyme activity. The frequency of in vivo TGr B cells was estimated to be 8.6-13.1 X 10(-6) for the 4 individuals by comparing the cloning efficiency of non-selected cells and TG-selected cells. This frequency is somewhat higher but comparable to the in vivo frequency of TGr T cells. Because the cloned TGr B cells can be easily expanded in vitro, this procedure provides a large amount of material for the precise characterization of in vivo mutations in humans.     

Preliminary studies on scoring micronuclei by computerised image analysis

Initial studies of the use of computerised image analysis to determine micronucleus frequencies in human lymphocytes that have completed one nuclear division are described. Two methods, based on (a) bromodeoxyuridine incorporation and (b) cytokinesis blocking with cytochalasin-B, were studied. The former method is directly amenable to automation. Cytokinesis-blocked cells could not be automatically recognised by image analysis but it was possible to obtain the correct micronucleus frequency from the integrated optical density histograms by using the mononucleate/binucleate cell ratio obtained by visual analysis. The mean (+/- 1 S.E.) integrated optical density of X-ray-induced micronuclei was 11.2% (+/- 1.1) of that measured for nuclei of G1 cells.     

The frequency of 6-thioguanine-resistant human peripheral blood lymphocytes as determined by flow cytometry and by clonal propagation

Human peripheral blood lymphocytes were selected for 6-thioguanine resistance in short-term cultures. Resistant cells, defined as cells capable of incorporating tritiated thymidine under the selective conditions, were flow-cytometrically differentiated with respect to their DNA content. This was carried out by sorting at two stages of the cell cycle, before and after mid-S-stage, yielding frequencies of resistant cells in the range of 10(-4) and 10(-5), respectively. Observed frequencies for cells from the whole cell cycle spectrum and for cells cultured according to the long-term protocol, the clonal assay, were in the range of 10(-4) and 10(-5), respectively. Our interpretation of these results is that the over-representation of tritiated thymidine-labelled cells occurring before mid-S-stage after short-term culture reflects less resistant cells or phenocopies which are probably eliminated during long-term culture with the clonal assay, hence leading to a decreased frequency of 6-thioguanine-resistant cells. We conclude, therefore, that short-term culture in combination with flow cytometric sorting after mid-S-stage in the cell cycle can be used as an alternative to the clonal assay for the determination of fully 6-thioguanine-resistant human peripheral lymphocytes.     

Problems and pitfalls in assessing human T-lymphocyte mutant frequencies

The measurement of 6-thioguanine-resistant frequencies in human T-lymphocytes has been used to quantitate the in vivo HPRT mutant frequency. The data so far indicate a large variability in normal healthy individuals. The reliability with which wells are identified for clonal growth in the assay was investigated using 5 different methods of scoring: visual scoring, uptake of [3H]thymidine (either by cut off point or by statistical analysis), cell count and cytogenetic analysis. None of these methods presented a viable means of scoring the assay. An examination of the statistical precision of the assay under the limitations imposed by the experimental conditions leads to the conclusion that there is a large inherent error associated with the estimated mutant frequencies. Analysis of the T-lymphocyte subpopulations by cell surface monoclonal antibodies also leads us to believe that the observed mutant frequencies may not be representative of the true in vivo mutant frequencies. If the assay is to be used as a sensitive screen for individual or population exposure to possible mutagens, a closer understanding of the biology of the assay is indicated, and a comprehensive reevaluation of the methodology required. The utility of the system for studying qualitative aspects of human mutagenesis is not in doubt.     

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

Inducibility of 6-thioguanine-resistant (6TGr) mutants and single-strand scission of DNA by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Frequency of 6TGr mutants increased concentration dependently by 24-h treatment with CdCl2 up to 3 X 10(-6) M but decreased beyond 3 X 10(-6) M. Mutagenic potency of cadmium in the absence of S9 was about half that of benzo[a]pyrene in the presence of S9 at equitoxic concentrations. Treatment of the cultured cells with cadmium after benzo[a]pyrene treatment was not synergistic but additive to the mutagenicity of benzo[a]pyrene. Single-strand scission of DNA by alkaline elution techniques was observed in the cells treated with CdCl2 for 2 h in a concentration-dependent manner. The single-strand scission by cadmium was detected only in combination with proteinase K digestion of the cell lysates, indicating formation of DNA--protein cross-linking by the metal. These biological and biochemical findings indicate that cadmium is mutagenic in mammalian cells, and its mutagenic effect seems to be accompanied by single-strand scission of DNA.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.     

International Commission for the Protection of the Environment against Mutagens and Carcinogens: a historical perspective

Hypoxanthine guanine phosphoribosyl transferase (HPRT) deficient human peripheral blood lymphocytes are usually enumerated either by the cloning assay or by the autoradiographic short-term assay. The short-term approach presented here is based on flow cytometric (FCM) scoring of 6-thioguanine (6-TG) resistant lymphocytes. HPRT-variants are enumerated on the basis of both DNA synthesis (by use of immunofluorescent detection of incorporated 5-bromo-2-deoxyuridine, BrdU) and total DNA content (by propidium iodide (PI) incorporation) of proliferating cells, i.e. the cells must both be labelled with BrdU and reside in late-S or G2 phase in order to be scored as a HPRT-variant. This approach is combined with a stringent discrimination of false-positive events, minimising occurrence of phenocopies or other non-specifically labelled cells that might falsely be scored as true HPRT-variants. The HPRT-variant frequency (V(f)) found by the presented method varied between 0.8 x 10(-5) and 5.8 x 10(-5) for healthy male and female donors aged between 20 and 74 years. There was no significant gender difference in V(f). A strong linear correlation was found between HPRT-variant frequency and age, showing an increase of 0.56 x 10(-6) per year of age (r(2)=0.62, P<0.001). The frequencies of false-positive events found showed a mean of 0.22 x 10(-5) in comparison with a pooled mean V(f) of 2.87 x 10(-5). There was no significant age effect on the frequency of false events (r(2)=0.15, P<0.095). The method presented here may provide a rapid and sensitive alternative to the autoradiographic technique for the short-term enumeration of HPRT-variants.     

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol

Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TGr) and at the Na/K-ATPase locus to ouabain resistance (OuaR). Treatment was for 24 h at 37 degrees C. TCHQ produced statistically significant increases in the frequency of TGr mutants. The lowest observed effective dose (LOED) was 20 microM, where the relative cloning efficiency was 63%. The relationship between the dose of TCHQ and the frequency of TGr mutants was approximately linear over the range of 0-60 microM with an estimated slope (+/- 95% confidence limits) of 1.1 +/- 0.3 mutants per 10(6) clonable cells per microM. At the highest tested dose of TCHQ, 60 microM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce TGr mutants at doses up to 120 microM. The relative cloning efficiency at this dose was 5%. Both TCHQ and TCC were unable to induce OuaR mutants. The results suggest that TCHQ is at least partly responsible for the genotoxic activity of pentachlorophenol. TCHQ can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to TGr but not to OuaR.