Humans are environmentally and occupationally exposed to polycyclic aromatic hydrocarbons (PAH). ... more Humans are environmentally and occupationally exposed to polycyclic aromatic hydrocarbons (PAH). PAH's are a class of tumorigenic compounds which act through metabolic transformation to chemically reactive forms, epoxides, which covalently bind to DNA forming DNA adducts. To evaluate the genotoxic effects of PAH's, air and urine samples were analyzed for PAH. Blood samples were analyzed for benzo(a)pyrene-diol-epoxide-DNA (BPDE-DNA) adducts. New methods for analyzing DNA adducts in lymphocytes have been used to study the genotoxic effects of human exposure to carcinogens. BPDE-DNA adducts in lymphocytes have been used as internal dosimeters of exposure to PAH's and several studies have been conducted. We measured BPDE-DNA adducts in aluminium plant workers with immuno-assay and physico-chemical methods. PAH-DNA adducts were detectable to a lesser extent in subjects working in an aluminium plant compared to subjects working in a coke oven plant.
Five monoclonal antibodies were obtained after fusion of mouse P3 x 63 myeloma cells with spleen ... more Five monoclonal antibodies were obtained after fusion of mouse P3 x 63 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin B1-adducted DNA complexes with methylated bovine serum albumin. Selected hybridomas were found to produce monoclonal antibodies specific for modified DNA containing both the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1, suggesting that these DNA adducts share a common antigenic determinant. Enzyme immunoassay conditions using these monoclonal antibodies were optimized, and DNA isolated from the livers of rats given dosages of aflatoxin B1 ranging from 0.01 to 1.0 mg aflatoxin B1 per kg body weight was tested. A level of modification in DNA of 1 aflatoxin B1 residue per 1,355,000 nucleotides can be quantitatively measured. Monoclonal antibodies will be useful probes for studying the molecular interactions of aflatoxin B1 with DNA and the occurrence of aflatoxin B1:DNA adducts in tissues and cells of humans exposed to this environmental carcinogen.
Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were quantitatively determined by ultrasensitiv... more Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were quantitatively determined by ultrasensitive radioimmunoassay (USERIA) and 32P postlabeling in 128 DNA samples from WBCs of 68 coke oven workers and a local control group of 13 workers. Forty-four samples had a detectable adduct level by USERIA, with a mean of 0.390 fmol adducts/micrograms DNA (12.9 adducts/10(8) nucleotides) in the exposed group compared to a mean of 0.316 fmol adducts/micrograms DNA (10.4 adducts/10(8) nucleotides) in the control group. The mean adduct level with 32P postlabeling was 0.05 fmol/micrograms DNA (1.67 adducts/10(8) nucleotides) for the exposed group and 0.046 fmol/microgram DNA (1.54 adducts/10(8) nucleotides for the control group. Based on job description the workers were divided in 4 groups: control, low-, medium-, and high-exposure group. Both methods produced a positive correlation coefficient between estimated exposure and PAH-DNA adduct levels. The significance levels determined with Kendall rank correlation were P = 0.0145 for USERIA and P = 0.0594 for 32P postlabeling. Adduct levels determined by 32P postlabeling showed a correlation with tobacco smoking in the control group. No significant correlation between PAH-DNA adduct levels measured by USERIA and 32P postlabeling was found. These results show that these methods recognize different parts of the complex exposures in a coke oven plant.
Application of methods for the measurement of DNA and protein adducts in environmental studies wa... more Application of methods for the measurement of DNA and protein adducts in environmental studies was surveyed. The methods included the 32P-postlabelling assay, immunoassay and synchronous fluorescence spectroscopy for DNA adducts. Additionally, methods for detecting excreted urinary RNA and DNA adducts were discussed. The protein adduct techniques included both immunological and chemical assays. The techniques have been applied in occupational and environmental studies, but usually one assay at a time. As specific DNA adducts can now be assayed for, it would be important to use these methods and specific protein adduct assays in the same studies. It is important to develop further specific adduct tests. This can be done with the help of standard compounds, which also allow quantitation in the assays. An international bank of standard compounds would be a major advancement to human biomonitoring.
The ability to repair damaged DNA was determined in different cell populations of rabbit lung cel... more The ability to repair damaged DNA was determined in different cell populations of rabbit lung cells isolated by centrifugal elutriation. DNA excision repair, measured as unscheduled DNA synthesis, was examined in in vitro confluent primary cultures. A dose dependent level of DNA excision repair was observed in alveolar type II cells after exposure to the direct acting alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and methyl methansesulphonate. Furthermore, O6-alkylguanine-DNA alkyltransferase activity was easily detectable in alveolar type II cells and alveolar macrophages. In contrast, non-ciliated (Clara) cells had 4 to 20-fold lower levels of DNA excision repair and non-detectable levels of O6-alkylguanine-DNA alkyltransferase. Uracil-DNA glycosylase activities in Clara cells and alveolar type II cells were in the same range and had 3-fold lower activity than alveolar macrophages. Our findings indicate that various lung cells differ in DNA repair capacity and may thus differ in sensitivity to some carcinogens.
Synchronous scanning fluorescence with a fixed wavelength difference (delta lambda) of 34 nm betw... more Synchronous scanning fluorescence with a fixed wavelength difference (delta lambda) of 34 nm between excitation and emission was used to quantitate benzo[a]pyrene-diol epoxide (BPDE)-DNA adducts. Fluorescence emission maxima occurred at 382 nm for BPDE-DNA and at 379 nm for benzo[a]pyrene-tetrols and -triol, which are hydrolysis products of BPDE. Similarly, the peak for pyrene was at 372 nm and for 1-nitropyrene at 386 nm. The minimum detectable amount of BPDE-moieties in in vitro modified BPDE-DNA, after hydrolysis in HCl, was 20 fmol in 100 micrograms of DNA, which is equivalent to 1 adduct per 1.4 X 10(7) nucleotides. The correlation of fluorescence intensity and the amount of BPDE-moieties was linear between 20 fmol and 1 pmol. DNA isolated from human lymphoblasts incubated with BPDE had the same fluorescence peak and the correlation between the fluorescence intensity and the amount of BPDE in the incubation mixture was linear. Among the DNA-samples from peripheral blood lymphocytes of 30 aluminum plant workers, only one sample was found to contain a peak similar to BPDE-DNA. None of the DNA-samples from 10 persons not occupationally exposed were positive. Measurement of BPDE-DNA adducts by synchronous fluorescence spectrophotometry should be useful in monitoring human exposure to benzo[a]pyrene.
Nickel is a toxic metal of environmental concern that has been found to be carcinogenic in man an... more Nickel is a toxic metal of environmental concern that has been found to be carcinogenic in man and animals. Primary human kidney (NHKE) cells were immortalized or rescued from senescence after exposure to NiSO4. The cell lines (IHKE) displayed abnormal karyotype and anchorage independent growth was observed. However, none of the IHKE cells produced tumor upon injection into athymic nude mice. Transfer of the v-Ha-ras oncogene into IHKE cells induced conversion of the immortalized cells into cell lines (THKE) that were tumorigenic when transplanted into athymic nude mice. Ha-ras DNA was present in the transformed cell lines and expressed at high level.
Chronic obstructive disease (COPD) is characterized by irreversible airflow obstruction and is as... more Chronic obstructive disease (COPD) is characterized by irreversible airflow obstruction and is associated with chronic local and systemic inflammation and oxidative stress. The enhanced oxidative stress and inflammation have been reported affect telomere length (TL). Furthermore, a number of SNPs at loci encoding the main components of the telomerase genes, TERT and TERC have been shown to correlate with TL. We aimed to explore the leukocyte TL and genotypes for single nucleotide polymorphisms, rs12696304 (C>G) and rs10936599 (C>T) near TERC in COPD cases and controls using q-PCR technologies. Successful assessment of TL was performed for 91 patients and 88 controls. The patients had shorter TL (17919.36±1203.01 bp) compared to controls (21 271.48±1891.36 bp) although not significant (p=0.137). The TL did not associate with the gender, age, spirometric indexes but correlated positively with the smoking habits (packs-years,Rho=0.422, p=0.032) and tended to correlate negatively with BMI (Rho= -0.215, p=0.076) in the controls, but not in COPD patients. The genotype frequencies of the SNPs rs12696304 and rs10936599 were compared between patients and controls and the odds ratios for developing COPD were calculated. The carriers of the common homozygous genotype of the SNPs had higher risk for COPD, compared to carriers of the variants alleles (rs12696304 CG+GG vs CC; OR=1.39, 95% CI:0.95-2.04, p=0.098 and for rs10936599 CT+TT vs CC OR=1.50, 95% CI:1.03-2.19, p=0.044). There was no association between the SNP genotypes and TL. In summary, our results suggest that COPD patients may have shorter TL, and rs12696304 and rs10936599 near TERC may affect the risk of COPD independently of TL.
In the previous monograph the chapter on exposure monitoring covered air and biological sampling ... more In the previous monograph the chapter on exposure monitoring covered air and biological sampling [1]. An analysis was given of parent compounds and, in the case of biological monitoring, of their metabolites in the body. In the present chapter we discuss a new form of biological monitoring: measurement of covalent reaction products (adducts) of the carcinogen with DNA and protein. In addition, we discuss the consequences of the adducts, i.e., mutations. The examples are taken from environmental exposures.
The concept of multistage carcinogenesis was largely formulated by investigators observing the in... more The concept of multistage carcinogenesis was largely formulated by investigators observing the interactive effects of viruses and chemicals. Rous and his coworkers (Rous and Kidd 1938; Rous and Friedewald 1944) found that virally induced skin papillomas that had regressed could be made to reappear by chemical irritants and concluded that the induced growth of these latent or dormant tumor cells occurred by a different mechanism (tumor promotion) than tumor induction (tumor initiation). Berenblum and Shubik (1947) more precisely divided carcinogenesis into initiation and promotion stages, and today the development of cancer is considered to be a multistep process involving several genetic and epigenetic steps (Boutwell 1974; Cairns 1975). Viruses are able to act at various stages of carcinogenesis, and interactive effects between environmental chemical carcinogens and viruses may be of substantial importance in human carcinogenesis (Fig. 1). Viral infections are widespread in humans, and viruses are factors in several types of human cancer, e.g., Burkitt’s lymphoma, nasopharyngeal cancer, liver cancer, T-cell leukemia, skin cancer, and cervical cancer (for review, see Phillips 1983). The purpose of our review is to summarize the current status of laboratory and epidemiological studies designed to investigate interactive effects of viruses and chemicals in carcinogenesis (Table 1).
Humans are environmentally and occupationally exposed to polycyclic aromatic hydrocarbons (PAH). ... more Humans are environmentally and occupationally exposed to polycyclic aromatic hydrocarbons (PAH). PAH's are a class of tumorigenic compounds which act through metabolic transformation to chemically reactive forms, epoxides, which covalently bind to DNA forming DNA adducts. To evaluate the genotoxic effects of PAH's, air and urine samples were analyzed for PAH. Blood samples were analyzed for benzo(a)pyrene-diol-epoxide-DNA (BPDE-DNA) adducts. New methods for analyzing DNA adducts in lymphocytes have been used to study the genotoxic effects of human exposure to carcinogens. BPDE-DNA adducts in lymphocytes have been used as internal dosimeters of exposure to PAH's and several studies have been conducted. We measured BPDE-DNA adducts in aluminium plant workers with immuno-assay and physico-chemical methods. PAH-DNA adducts were detectable to a lesser extent in subjects working in an aluminium plant compared to subjects working in a coke oven plant.
Five monoclonal antibodies were obtained after fusion of mouse P3 x 63 myeloma cells with spleen ... more Five monoclonal antibodies were obtained after fusion of mouse P3 x 63 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin B1-adducted DNA complexes with methylated bovine serum albumin. Selected hybridomas were found to produce monoclonal antibodies specific for modified DNA containing both the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1, suggesting that these DNA adducts share a common antigenic determinant. Enzyme immunoassay conditions using these monoclonal antibodies were optimized, and DNA isolated from the livers of rats given dosages of aflatoxin B1 ranging from 0.01 to 1.0 mg aflatoxin B1 per kg body weight was tested. A level of modification in DNA of 1 aflatoxin B1 residue per 1,355,000 nucleotides can be quantitatively measured. Monoclonal antibodies will be useful probes for studying the molecular interactions of aflatoxin B1 with DNA and the occurrence of aflatoxin B1:DNA adducts in tissues and cells of humans exposed to this environmental carcinogen.
Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were quantitatively determined by ultrasensitiv... more Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were quantitatively determined by ultrasensitive radioimmunoassay (USERIA) and 32P postlabeling in 128 DNA samples from WBCs of 68 coke oven workers and a local control group of 13 workers. Forty-four samples had a detectable adduct level by USERIA, with a mean of 0.390 fmol adducts/micrograms DNA (12.9 adducts/10(8) nucleotides) in the exposed group compared to a mean of 0.316 fmol adducts/micrograms DNA (10.4 adducts/10(8) nucleotides) in the control group. The mean adduct level with 32P postlabeling was 0.05 fmol/micrograms DNA (1.67 adducts/10(8) nucleotides) for the exposed group and 0.046 fmol/microgram DNA (1.54 adducts/10(8) nucleotides for the control group. Based on job description the workers were divided in 4 groups: control, low-, medium-, and high-exposure group. Both methods produced a positive correlation coefficient between estimated exposure and PAH-DNA adduct levels. The significance levels determined with Kendall rank correlation were P = 0.0145 for USERIA and P = 0.0594 for 32P postlabeling. Adduct levels determined by 32P postlabeling showed a correlation with tobacco smoking in the control group. No significant correlation between PAH-DNA adduct levels measured by USERIA and 32P postlabeling was found. These results show that these methods recognize different parts of the complex exposures in a coke oven plant.
Application of methods for the measurement of DNA and protein adducts in environmental studies wa... more Application of methods for the measurement of DNA and protein adducts in environmental studies was surveyed. The methods included the 32P-postlabelling assay, immunoassay and synchronous fluorescence spectroscopy for DNA adducts. Additionally, methods for detecting excreted urinary RNA and DNA adducts were discussed. The protein adduct techniques included both immunological and chemical assays. The techniques have been applied in occupational and environmental studies, but usually one assay at a time. As specific DNA adducts can now be assayed for, it would be important to use these methods and specific protein adduct assays in the same studies. It is important to develop further specific adduct tests. This can be done with the help of standard compounds, which also allow quantitation in the assays. An international bank of standard compounds would be a major advancement to human biomonitoring.
The ability to repair damaged DNA was determined in different cell populations of rabbit lung cel... more The ability to repair damaged DNA was determined in different cell populations of rabbit lung cells isolated by centrifugal elutriation. DNA excision repair, measured as unscheduled DNA synthesis, was examined in in vitro confluent primary cultures. A dose dependent level of DNA excision repair was observed in alveolar type II cells after exposure to the direct acting alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and methyl methansesulphonate. Furthermore, O6-alkylguanine-DNA alkyltransferase activity was easily detectable in alveolar type II cells and alveolar macrophages. In contrast, non-ciliated (Clara) cells had 4 to 20-fold lower levels of DNA excision repair and non-detectable levels of O6-alkylguanine-DNA alkyltransferase. Uracil-DNA glycosylase activities in Clara cells and alveolar type II cells were in the same range and had 3-fold lower activity than alveolar macrophages. Our findings indicate that various lung cells differ in DNA repair capacity and may thus differ in sensitivity to some carcinogens.
Synchronous scanning fluorescence with a fixed wavelength difference (delta lambda) of 34 nm betw... more Synchronous scanning fluorescence with a fixed wavelength difference (delta lambda) of 34 nm between excitation and emission was used to quantitate benzo[a]pyrene-diol epoxide (BPDE)-DNA adducts. Fluorescence emission maxima occurred at 382 nm for BPDE-DNA and at 379 nm for benzo[a]pyrene-tetrols and -triol, which are hydrolysis products of BPDE. Similarly, the peak for pyrene was at 372 nm and for 1-nitropyrene at 386 nm. The minimum detectable amount of BPDE-moieties in in vitro modified BPDE-DNA, after hydrolysis in HCl, was 20 fmol in 100 micrograms of DNA, which is equivalent to 1 adduct per 1.4 X 10(7) nucleotides. The correlation of fluorescence intensity and the amount of BPDE-moieties was linear between 20 fmol and 1 pmol. DNA isolated from human lymphoblasts incubated with BPDE had the same fluorescence peak and the correlation between the fluorescence intensity and the amount of BPDE in the incubation mixture was linear. Among the DNA-samples from peripheral blood lymphocytes of 30 aluminum plant workers, only one sample was found to contain a peak similar to BPDE-DNA. None of the DNA-samples from 10 persons not occupationally exposed were positive. Measurement of BPDE-DNA adducts by synchronous fluorescence spectrophotometry should be useful in monitoring human exposure to benzo[a]pyrene.
Nickel is a toxic metal of environmental concern that has been found to be carcinogenic in man an... more Nickel is a toxic metal of environmental concern that has been found to be carcinogenic in man and animals. Primary human kidney (NHKE) cells were immortalized or rescued from senescence after exposure to NiSO4. The cell lines (IHKE) displayed abnormal karyotype and anchorage independent growth was observed. However, none of the IHKE cells produced tumor upon injection into athymic nude mice. Transfer of the v-Ha-ras oncogene into IHKE cells induced conversion of the immortalized cells into cell lines (THKE) that were tumorigenic when transplanted into athymic nude mice. Ha-ras DNA was present in the transformed cell lines and expressed at high level.
Chronic obstructive disease (COPD) is characterized by irreversible airflow obstruction and is as... more Chronic obstructive disease (COPD) is characterized by irreversible airflow obstruction and is associated with chronic local and systemic inflammation and oxidative stress. The enhanced oxidative stress and inflammation have been reported affect telomere length (TL). Furthermore, a number of SNPs at loci encoding the main components of the telomerase genes, TERT and TERC have been shown to correlate with TL. We aimed to explore the leukocyte TL and genotypes for single nucleotide polymorphisms, rs12696304 (C>G) and rs10936599 (C>T) near TERC in COPD cases and controls using q-PCR technologies. Successful assessment of TL was performed for 91 patients and 88 controls. The patients had shorter TL (17919.36±1203.01 bp) compared to controls (21 271.48±1891.36 bp) although not significant (p=0.137). The TL did not associate with the gender, age, spirometric indexes but correlated positively with the smoking habits (packs-years,Rho=0.422, p=0.032) and tended to correlate negatively with BMI (Rho= -0.215, p=0.076) in the controls, but not in COPD patients. The genotype frequencies of the SNPs rs12696304 and rs10936599 were compared between patients and controls and the odds ratios for developing COPD were calculated. The carriers of the common homozygous genotype of the SNPs had higher risk for COPD, compared to carriers of the variants alleles (rs12696304 CG+GG vs CC; OR=1.39, 95% CI:0.95-2.04, p=0.098 and for rs10936599 CT+TT vs CC OR=1.50, 95% CI:1.03-2.19, p=0.044). There was no association between the SNP genotypes and TL. In summary, our results suggest that COPD patients may have shorter TL, and rs12696304 and rs10936599 near TERC may affect the risk of COPD independently of TL.
In the previous monograph the chapter on exposure monitoring covered air and biological sampling ... more In the previous monograph the chapter on exposure monitoring covered air and biological sampling [1]. An analysis was given of parent compounds and, in the case of biological monitoring, of their metabolites in the body. In the present chapter we discuss a new form of biological monitoring: measurement of covalent reaction products (adducts) of the carcinogen with DNA and protein. In addition, we discuss the consequences of the adducts, i.e., mutations. The examples are taken from environmental exposures.
The concept of multistage carcinogenesis was largely formulated by investigators observing the in... more The concept of multistage carcinogenesis was largely formulated by investigators observing the interactive effects of viruses and chemicals. Rous and his coworkers (Rous and Kidd 1938; Rous and Friedewald 1944) found that virally induced skin papillomas that had regressed could be made to reappear by chemical irritants and concluded that the induced growth of these latent or dormant tumor cells occurred by a different mechanism (tumor promotion) than tumor induction (tumor initiation). Berenblum and Shubik (1947) more precisely divided carcinogenesis into initiation and promotion stages, and today the development of cancer is considered to be a multistep process involving several genetic and epigenetic steps (Boutwell 1974; Cairns 1975). Viruses are able to act at various stages of carcinogenesis, and interactive effects between environmental chemical carcinogens and viruses may be of substantial importance in human carcinogenesis (Fig. 1). Viral infections are widespread in humans, and viruses are factors in several types of human cancer, e.g., Burkitt’s lymphoma, nasopharyngeal cancer, liver cancer, T-cell leukemia, skin cancer, and cervical cancer (for review, see Phillips 1983). The purpose of our review is to summarize the current status of laboratory and epidemiological studies designed to investigate interactive effects of viruses and chemicals in carcinogenesis (Table 1).
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