Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

Cumulative exposure assessment for trace-level polycyclic aromatic hydrocarbons (PAHs) using human blood and plasma analysis

Humans experience chronic cumulative trace-level exposure to mixtures of volatile, semi-volatile, and non-volatile polycyclic aromatic hydrocarbons (PAHs) present in the environment as by-products of combustion processes. Certain PAHs are known or suspected human carcinogens and so we have developed methodology for measuring their circulating (blood borne) concentrations as a tool to assess internal dose and health risk. We use liquid/liquid extraction and gas chromatography–mass spectrometry and present analytical parameters including dynamic range (0–250 ng/ml), linearity (>0.99 for all compounds), and instrument sensitivity (range 2–22 pg/ml) for a series of 22 PAHs representing 2–6-rings. The method is shown to be sufficiently sensitive for estimating PAHs baseline levels (typical median range from 1 to 1000 pg/ml) in groups of normal control subjects using 1-ml aliquots of human plasma but we note that some individuals have very low background concentrations for 5- and 6-ring compounds that fall below robust quantitation levels.     

Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.     

多环芳烃的真菌漆酶转化及污染土壤修复技术

漆酶可以转化多种有机污染物,在环境保护领域具有广泛的应用潜力。二十年来,通过多学科协同研究,对真菌漆酶转化多环芳烃的机制、特征等各方面的认识不断深入。基于漆酶等真菌木质素分解酶的污染土壤修复技术不断发展,并逐渐走向田间应用。本文首先介绍了真菌漆酶的一般作用机制与多环芳烃转化特征,结合我们的相关研究提出了漆酶作用下多环芳烃在土壤中的迁移模式;其次介绍了利用漆酶氧化原理修复污染农田土壤的潜力,着重对利用农业废弃物进行真菌生物刺激的修复实践进行了评述;最后,就漆酶转化多环芳烃基础研究中的若干重要问题进行了思考,并展望了真菌及其漆酶系统在污染土壤修复应用中的发展方向。     

Biotreatability of polycyclic aromatic hydrocarbons in brackish sediments: Preliminary studies of an integrated monitoring

An integrated monitoring, of chemical, microbiological and ecotoxicological parameters, was performed for a biotreatability study of polycyclic aromatic hydrocarbons (PAHs)—contaminated brackish sediments. Three slurry reactors were prepared, consisting of (a) a slurry with sediment and seawater called TQ slurry, to evaluate the intrinsic bioremediation potential, (b) a slurry with the addition of a selected microbial consotrium called BIO slurry, to evaluate the bioaugmentation effect, (c) a slurry with the addition of Soya lecithin called LEC slurry, to evaluate the effect of the addition of a natural surfactant. Biodegradation results showed that both BIO and LEC slurries enhanced PAHs removal, increasing the biodegradation rate for 5- and 6-ring PAHs. Furthermore, ecotoxicological response (Microtox® assay on whole sediment, aqueous extract and organic extract) demonstrated a detoxification of the PAHs initial mixture only for BIO slurry. The findings that aerobic PAHs degradation can be stimulated via inoculation with adapted sediment bacteria suggest that a bioaugmentation process may be a useful strategy for ex-situ treatment.     

Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.     

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 μg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 μg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods

Protein profiles of Mycobacterium vanbaalenii PYR-1 grown in the presence of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) were examined by two-dimensional gel electrophoresis (2-DE). Cultures of M. vanbaalenii PYR-1 were incubated with pyrene, pyrene-4,5-quinone (PQ), phenanthrene, anthracene, and fluoranthene. Soluble cellular protein fractions were analyzed and compared, using immobilized pH gradient (IPG) strips. More than 1000 gel-separated proteins were detected using a 2-DE analysis program within the window of isoelectric point (pI) 4-7 and a molecular mass range of 10-100 kDa. We observed variations in the protein composition showing the upregulation of multiple proteins for the five PAH treatments compared with the uninduced control sample. By N-terminal sequencing or mass spectrometry, we further analyzed the proteins separated by 2-DE. Due to the lack of genome sequence information for this species, protein identification provided an analytical challenge. Several PAH-induced proteins were identified including a catalase-peroxidase, a putative monooxygenase, a dioxygenase small subunit, a small subunit of naphthalene-inducible dioxygenase, and aldehyde dehydrogenase. We also identified proteins related to carbohydrate metabolism (enolase, 6-phosphogluconate dehydrogenase, indole-3-glycerol phosphate synthase, and fumarase), DNA translation (probable elongation factor Tsf), heat shock proteins, and energy production (ATP synthase). Many proteins from M. vanbaalenii PYR-1 showed similarity with protein sequences from M. tuberculosis and M. leprae. Some proteins were detected uniquely upon exposure to a specific PAH whereas others were common to more than one PAH, which indicates that induction triggers not only specific responses but a common response in this strain.     

Investigating tea temperature and content as risk factors for esophageal cancer in an endemic region of Western Kenya: Validation of a questionnaire and analysis of polycyclic aromatic hydrocarbon content

BackgroundEsophageal squamous cell carcinoma (ESCC) is common in certain areas worldwide. One area, western Kenya, has a high risk of ESCC, including many young cases (<30 years old), but has limited prior study of potential risk factors. Thermal injury from hot food and beverages and exposure to polycyclic aromatic hydrocarbons (PAHs) have been proposed as important risk factors for ESCC in other settings. The beverage of choice in western Kenya is milky tea (chai).MethodsHealthy individuals >18 years of age who were accompanying relatives to an endoscopy unit were recruited to participate. The preferred initial temperature of chai consumption in these adults was measured by questionnaire and digital thermometer. Comparisons of these results were assessed by kappa statistics. Concentrations of 26 selected PAHs were determined by gas chromatography/mass spectrometry in samples of 11 brands of commercial tea leaves commonly consumed in Kenya.ResultsKappa values demonstrated moderate agreement between questionnaire responses and measured temperatures. The mean preferred chai temperatures were 72.1 °C overall, 72.6 °C in men (n = 78), and 70.2 °C in women (n = 22; p < 0.05). Chai temperature did not significantly differ by age or ethnic group. The PAH levels in the commercial Kenyan tea leaves were uniformly low (total PAH < 300 ng/g of leaves).ConclusionsStudy participants drink chai at higher temperatures than previously reported in other high-risk ESCC regions. Chai is not, however, a source of significant PAH exposure. Very hot chai consumption should be further evaluated as a risk factor for ESCC in Kenya with the proposed questionnaire.     

Prenatal exposure to polycyclic aromatic hydrocarbons could increase the risk of low birth weight by affecting the DNA methylation states in a Chinese cohort

Polycyclic aromatic hydrocarbons (PAHs), as a kind of endocrine disruptors, can enter the fetus body cross the placental barrier from prenatal PAHs exposure to cause adverse birth outcomes. However, it is controversial association between prenatal PAHs exposure and low birth weight (LBW) of their infants. So the present study aimed to estimate the effects of prenatal PAHs exposure during the pregnancy on the risk of LBW in a Chinese cohort through modifying the DNA methylation states. A longitudinal prospective study with 407 pregnant women was established from May to October 2019. The prenatal PAHs exposure during the pregnancy was assessed using the internal dose such as the PAHs metabolites and PAH-DNA adducts in the umbilical cord blood. The methylation levels of genomic DNA and growth-related genes (IGF1 and IGF2) were assessed, while the expressions of these genes were both determined by RT-PCR and Elisa methods. The growth outcomes and relevant Z-scores were recorded at birth. The correlations between the DNA methylation status and concentrations of PAHs, expression levels of growth-related genes and body weight/WAZ were investigated as the measures. According to the PAH-DNA adducts, the subjects were divided into two groups: PAHs-exposed group (PAH-DNA adducts>0, n = 55) and non-exposed group (PAH-DNA adducts = 0, n = 352). Compared with the non-exposed group, it displayed marked decreased birth weight, and increased concentrations of PAHs and DNA methylation levels of the global genomic, IGF1 and IGF2 with their lower expressions in the PAHs-exposed group. These hypermethylation (global genomic, CpG14 and CpG15 of IGF1, and CpG14 of IGF2) were positively correlated with the contents of PAHs in the umbilical cord blood, and negatively correlated with the growth outcomes and their expressions. Totally, prenatal PAHs exposures may contribute to an increased risk of LBW of their infants by modulating the DNA methylation states of genomic DNA and growth-related genes (IGF1 and IGF2) in the umbilical cord blood, which could provide the prenatal prevention of PAHs exposure from possible environmental media except from the occupation and tobacco usage to ensure the health of their infants.     

Characteristic expression of aryl hydrocarbon receptor repressor gene in human tissues: organ-specific distribution and variable induction patterns in mononuclear cells

To investigate the expression of aryl hydrocarbon receptor repressor (AhRR) and related molecules in various tissues and the effects of aromatic hydrocarbons (AHs) on their expression, we developed a reliable technique of quantification of human AhRR as well as aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT) and cytochrome P450 1A1 (CYP1A1) mRNA by real-time TaqMan PCR method. First, we examined the expression of these genes in human adult or fetal tissues. The levels of AhRR expression were extremely high in testis, very high in lung, ovary, spleen and pancreas from adults, whereas those were low in those from fetuses. On the other hand, CYP1A1 expression was extremely high in lung, and AhR and ARNT were ubiquitously expressed in almost all tissues. Second, we compared the expression levels of these genes in mononuclear cells (MNCs) from various sources. Comparison of the basal expression levels of these genes in MNCs demonstrated that MNCs from umbilical cord blood showed higher AhRR or CYP1A1 expression than those from adults. The induction of AhRR or CYP1A1 expression by 3-methylcholanthrene (3-MC) was observed in MNCs from adults but not from umbilical cord blood. Consequently, there existed characteristic differences in the basal levels of AhRR and CYP1A1 expression in MNCs, as well as in their inducibility by 3-MC among MNCs from various types of human bloods. These results will provide basic information for a possible application of AhRR and CYP1A1 measurements to evaluate AH exposure in vivo.     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

1-Methoxypyrene and 1,6-dimethoxypyrene: two novel metabolites in fungal metabolism of polycyclic aromatic hydrocarbons

The metabolism of pyrene by Penicillium glabrum strain TW 9424, a strain isolated from a site contaminated with polycyclic aromatic hydrocarbons (PAHs) was investigated in submerged cultures. The metabolites formed were identified as 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene, 1,6- and 1,8-pyrenequinone, and 1-pyrenyl sulfate. In addition, two new metabolites were isolated and identified by UV, ¹H nuclear magnetic resonance, and mass spectroscopy as 1-methoxypyrene and 1,6-dimethoxypyrene. Experiments with [methyl-³H]S-adenosyl-l-methionine (SAM) revealed that SAM is the coenzyme that provides the methyl group for the methyltransferase involved. To our knowledge, this is the first time that methoxylated metabolites of PAHs have been isolated from fungal cultures. Received: 27 August 1996 / Accepted: 8 January 1997     

The concentration of polycyclic aromatic hydrocarbons (PAHs) in mother milk: A global systematic review,meta-analysis and health risk assessment of infants

BackgroundBio-monitoring of polycyclic aromatic hydrocarbons (PAHs) contaminants in mother milk is essential to keep mothers and infants healthy against potential risks. The current study assesses the concentration of PAHs in mother milk through a meta-analytic and systematic review approach.MethodsAll the published studies up to December 2020 regarding the concentrations of various PAHs in mother milk were searched throughout major international databases such as PubMed, Scopus, and Web of Science. Moreover, the possible carcinogenic and mutagenic risks to infants were evaluated based on the BaP (benzo[a]pyrenee) equivalent dose.ResultsAccording to the results of 13 articles included among 936 retrieved studies, the lowest and highest concentration of PAHs was (0.125 ng/g) and (76.36 ng/g) related to benz(a)anthracenem and 1-methylnaphthalene, respectively. The highest (9.830 ng/g) and lowest (0.009 ng/g) concentration of PAHs was related to Mexico and Japan, respectively. Besides, carcinogenetic and mutagenic risk assessment of the PAHs indicated that risk pattern was different across countries. It can be concluded that the consumption of mother milk is safe and does not pose a risk due to the ingestion of PAHs to the health of infant consumers.     

Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity.

In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.     

Urinary 1-hydroxypyrene as a biomarker of exposure to polycyclic aromatic hydrocarbons: biological monitoring strategies and methodology for determining biological exposure indices for various work environments

This article reviews the published studies on urinary 1-hydroxypyrene (1-OHP) as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) in work environments. Sampling and analysis strategies as well as a methodology for determining biological exposure indices (BEIs) of 1-OHP in urine for different work environments are proposed for the biological monitoring of occupational exposure to PAHs. Owing to the kinetics of absorption of pyrene by different exposure routes and excretion of 1-OHP in urine, in general, 1-OHP urinary excretion levels increase during the course of a workday, reaching maximum values 3-9 h after the end of work. When the contribution of dermal exposure is important, post-shift 1-OHP excretion can however be lower than pre-shift levels in the case where a worker has been exposed occupationally to PAHs on the day prior to sampling. In addition, 1-OHP excretion levels in either pre-shift, post-shift or evening samples increase during the course of a work-week, levelling off after three consecutive days of work. Consequently, ideally, for a first characterization of a work environment and for an indication of the major exposure route, considering a 5-day work-week (Monday to Friday), the best sampling strategy would be to collect all micturitions over 24 h starting on Monday morning. Alternatively, collection of pre-shift, post-shift and evening urine samples on the first day of the work-week and at the end of the work-week is recommended. For routine monitoring, pre-shift samples on Monday and post-shift samples on Friday should be collected when pulmonary exposure is the main route of exposure. On the other hand, pre-shift samples on Monday and Friday should be collected when the contribution of skin uptake is important. The difference between beginning and end of work-week excretion will give an indication of the average exposure over the workweek. Pre-shift samples on the first day of the work-week will indicate background values, and, hence, reflect general environment exposure and body burden of pyrene and/or its metabolites. On the other hand, since PAH profile can vary substantially in different work sites, a single BEI cannot apply to all workplaces. A simple equation was therefore developed to establish BEIs for workers exposed to PAHs in different work environments by using a BEI already established for a given work environment and by introducing a correction factor corresponding to the ratio of the airborne concentration of the sum of benzo(a)pyrene (BaP) equivalent to that of pyrene. The sum of BaP equivalent concentrations represents the sum of carcinogenic PAH concentrations expressed as BaP using toxic equivalent factors. Based on a previously estimated BEI of 2.3 μmol 1-OHP mol^-1 creatinine for coke-oven workers, BEIs of 4.4, 8.0 and 9.8 μmol 1-OHP mol^-1 creatinine were respectively calculated for vertical pin Söderberg workers, anode workers and pre-bake workers of aluminium plants and a BEI of 1.2 μmol 1-OHP mol^-1 creatinine was estimated for iron foundry workers. This approach will allow the potential risk of cancer in individuals occupationally exposed to PAHs to be assessed better.     

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: a rapid method for its analysis using hydroxylapatite column chromatography.

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.