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N -Nitrosamines are contaminants found throughout the environment, including in drinking water, and many nitrosamines are likely potent carcinogens. Correspondingly, there is a need for rapid and cost-effective in-field detection methods that can provide timely information about their contamination levels in water. This study details a colorimetric assay for detecting aqueous N -nitrosodimethylamine (NDMA) by photochemical nitrosation of a commercial naphtholsulfonate, to offer an attractive alternative to traditional laboratory-based analysis. The resulting naphthoquinone-oxime coordinates to aqueous iron(II) ions to form a green complex, allowing for direct visual detection. Characterization via Mössbauer and electron paramagnetic resonance (EPR) spectroscopy, alongside single-crystal structure determination, provides comprehensive structure information on the iron indicator complex. Optimization of detection conditions, including UV irradiation and response times, led to an improved colorimetric detection method with a limit of detection of 0.66 ppm for NDMA. The practical applicability and selectivity of this colorimetric detection scheme make it a promising candidate for the development of field-deployable sensors for NDMA in environmental water samples.

The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C → T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gpt Δ C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG → TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA → CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development., Competing Interests: Competing interests statement:The patent US9982009 was granted to B.I.F., R.G.C., J.M.E., S.V.F., and S.S., and assigned to Massachusetts Institute of Technology and Yale University. The patent covers the use of the 11beta-dipropyl and related compounds as therapeutics for polycystic kidney disease and polycystic liver disease. All other authors declare no competing interests.

DNA-methylating environmental carcinogens such as N -nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O 6 -methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N -methyl- N -nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Cancer.)

Cu(I) from tetrakis(acetonitrile)copper(I) hexafluorophosphate ([Cu(MeCN) 4 ]PF 6 ) was complexed with five structurally related phosphines containing N-heterocycles. The interactions between the resulting complexes and some N -nitrosamines were studied using X-ray crystallography as well as emission spectroscopy. Upon complexation, three phosphine ligands bridge two Cu(I) centers to give paddlewheel type structures that displayed a range of emission wavelengths spanning the visible region. N -Nitrosodimethylamine (NDMA) was shown to coordinate to one of the two copper centers in some of the paddlewheel complexes in the solid state and this interaction also quenches their emissions in solution. The influence of the weakly coordinating anion on crystal and spectroscopic properties of one of the paddlewheel complexes was also examined using tetrakis(acetonitrile)copper(I) perchlorate ([Cu(MeCN) 4 ]ClO 4 ) as an alternative Cu(I) source. Similarly, copper(II) perchlorate hexahydrate (Cu(ClO 4 ) 2 ·6H 2 O) was used for complexation to observe the impact of metal oxidation state on the two aforementioned properties. Lastly, the spectroscopic properties of the complex between Ph 2 P(1-Isoquinoline) and Cu(I) was shown to exhibit solvent dependence when the counterion is ClO 4 - . These Cu(I) complexes are bench stable solids and may be useful materials for developing a fluorescence based detection method for N -nitrosamines.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N -Nitrosamines are found in food, drugs, air, water, and soil. They pose a significant risk to human health because of their carcinogenicity; consequently, materials that can be used to selectively and sensitively detect nitrosamines are needed. In this work, we designed and synthesized two polymers bearing calix[4]arene or 4- tert -butylcalix[4]arene tungsten-imido complexes (PCalixH and PCalixtBu) as N -nitrosodimethylamine (NDMA) receptors. The interaction between metallocalix[4]arene monomers/polymers and NDMA was confirmed by 1 H NMR and IR spectroscopy. Single-crystal X-ray analysis further revealed that the host-guest interaction is based on binding of the terminal oxygen of NDMA to tungsten within the calixarene cavity. Gravimetric detection of NDMA was performed on a quartz crystal microbalance (QCM) in air. Both polymers show responses to NDMA, with PCalixtBu exhibiting a low theoretical limit of detection of 5 ppb for NDMA. The sensor also shows high selectivity toward NDMA and moderate humidity tolerance. This work provides a sensitive sensor for detection of NDMA and also offers a class of new, selective, and efficient NDMA receptors for the future design of NDMA sensors and NDMA extraction materials.

N-Nitrosodimethylamine (NDMA) is a DNA-methylating agent that has been discovered to contaminate water, food, and drugs. The alkyladenine DNA glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we study Aag-knockout (Aag -/- ) and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine [3MeA]) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switches from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrate a suite of molecular, cellular, and physiological analyses. We find that unrepaired 3MeA is somewhat toxic, but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tip the balance between blocks and breaks and thus dictate the disease consequences of DNA damage., Competing Interests: Declaration of interests S.L.A., D.K.T., and S.D.D. are employees of Litron Laboratories. Litron plans to sell kits for scoring micronucleated mouse hepatocytes via flow cytometry as described herein (In Vivo MicroFlow PLUS ML kits)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is O 6 -methylguanine (m6G), which base pairs with thymine during replication to cause GC → AT mutations. The gpt delta C57BL/6J mouse strain of Nohmi et al. ( Mol. Mutagen 1996 , 28 , 465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from gpt delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by N -methyl- N -nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental N -nitrosamines, such as N -nitrosodimethylamine and therapeutic agents such as Temozolomide. The DNA repair protein MGMT ( O 6 -methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of Temozolomide in cancer therapy. The gpt delta MEFs were treated with MNU to establish dose-dependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC → AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using O 6 -methyl- d 3 -guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation frequency seen in the same cells. This work establishes gpt delta MEFs as a versatile tool for probing mutagenesis by environmental and therapeutic agents and as a cell culture model in which chemical genetics can be used to determine the impact of DNA repair on biological responses to DNA damaging agents.

N -Nitrosamines are environmental genotoxicants that are widely encountered in air, water, and food. Contamination of indoor and outdoor air with N -nitrosamines has been reported on many occasions. Conventional detection of airborne N -nitrosamines requires sophisticated instrumentation, field sampling, and laboratory analysis. Herein, we report ultrasensitive carbon nanotube based chemiresistive sensors utilizing a cobalt(III) tetraphenylporphyrin selector element for the detection of N -nitrosamines. Concentrations as low as 1 ppb N -nitrosodimethylamine, N -nitrosodiethylamine, and N -nitrosodibutylamine were detected. We also demonstrate the integration of these sensors with a field deployable sensing node wherein the sensor response can be read online remotely.

Pregnancy is a complex physiological state, in which the metabolism of endogenous as well as exogenous agents is ostensibly altered. One exogenous agent of concern is the hepatocarcinogen aflatoxin B1 (AFB1), a foodborne fungal toxin, that requires phase I metabolic oxidation for conversion to its toxic and carcinogenic form, the AFB1-8,9-exo-epoxide. The epoxide interacts with cellular targets causing toxicity and cell death; these targets include the covalent modification of DNA leading to mutations that can initiate malignant transformation. The main detoxification pathway of the AFB1-epoxide involves phase II metabolic enzymes including the glutathione-S-transferase (GST) family. Pregnancy can modulate both phase I and II metabolism and alter the biological potency of AFB1. The present work investigated the impact of pregnancy on AFB1 exposure in mice. A single IP dose of 6 mg/kg AFB1 was administered to pregnant C57BL/6 J mice at gestation day 14 and matched non-pregnant controls. Pregnant mice accumulated 2-fold higher AFB1-N7-guanine DNA adducts in the liver when compared with nonpregnant controls 6 h post-exposure. Enhanced DNA adduct formation in pregnant animals paralleled elevated hepatic protein expression of mouse CYP1A2 and mouse homologs of human CYP3A4, phase I enzymes capable of bioactivating AFB1. Although phase II enzymes GSTA1/2 showed decreased protein expression, GSTA3, the primary enzymatic protection against the AFB1-epoxide, was unaffected at the protein level. Taken together, our results reveal that pregnancy may constitute a critical window of susceptibility for maternal health, and provide insight into the biochemical factors that could explain the underlying risks., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.)

Using duplex-consensus sequencing technology, we recently identified the characteristic high-resolution mutational spectrum of the liver carcinogen aflatoxin B 1 in a mouse model, many months before aflatoxin-induced tumors are detectable. The diagnostic power of this spectrum is then demonstrated by accurately identifying, among the sequenced human liver tumors, the subset of cancers associated with aflatoxin B 1 exposure.

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB 1 ) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB 1 -induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB 1 -deoxyguanosine adduct (AFB 1 -Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB 1 show significant increases in the levels of AFB 1 -Fapy-dG in Neil1 -/- vs. wild-type liver DNA. Further, Neil1 -/- mice are highly susceptible to AFB 1 -induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1 -/- The magnitude of this effect in Neil1 -/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB 1 -associated HCCs., Competing Interests: The authors declare no conflict of interest.

Aflatoxin B 1 (AFB 1 ) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB 1 -DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB 1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB 1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (C G C; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB 1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB 1 , and, as such, is an early detection metric for AFB 1 -induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.

It has become axiomatic that critical windows of susceptibility to genotoxins exist and that genetic damage in utero may be a trigger for later life cancers. Data supporting this critical window hypothesis are remarkably few. This study provides a quantitative bridge between DNA damage by the liver carcinogen aflatoxin B1 (AFB1 ) during prenatal development and the risk of later life genetic disease. AFB1 was given to pregnant C57BL/6J mice, carrying F1 gestation day 14 (GD14) embryos of the B6C3F1 genotype. Ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS) using aflatoxin-(15) N5 -guanine adduct standards afforded measurement of the AFB1 -N(7) -Gua and AFB1 -FAPY adducts 6-hr post dosing in liver DNA of mothers and embryos. A parallel cohort gave birth and the livers of the F1 were analyzed for mutations in the gpt gene at 3 and 10 weeks of age. The data revealed mutational spectra dominated by G:C to T:A mutations in both the mother and offspring that are characteristic of AFB1 and distinct from background. It was shown that adducts in GD14 embryos were 20-fold more potent inducers of mutagenesis than adducts in parallel-dosed adults. This sensitivity enhancement correlated with Ki67 staining of the liver, reflecting the proliferative potential of the tissue. Taken together, these data provide insight into the relative genetic risks of prenatal and adult exposures to AFB1 . Early life exposure, especially during the embryonic period, is strikingly more mutagenic than treatment later in life. Moreover the data provide a baseline against which risk prevention strategies can be evaluated., (© 2014 UICC.)

Aflatoxin B1 (AFB1) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB1-DNA adducts in AFB1-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB1 and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4h after AFB1 administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB1-induced hepatotoxicity. At 24h after AFB1 administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB1-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB1 hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule. Here, we show that synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules. To prove the generality of this phenomenon, we report three examples of heteropolymer-nanotube recognition complexes for riboflavin, L-thyroxine and oestradiol. In each case, the recognition was predicted using a two-dimensional thermodynamic model of surface interactions in which the dissociation constants can be tuned by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatiotemporal sensors based on modulation of the carbon nanotube photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.

A convergent synthesis of a novel estrogen receptor-targeted drug hybrid was developed based on structures of the potent anti-proliferative mitomycin C and the steroidal anti-estrogen RU 39411. The steroidal antiestrogen was prepared with an azido-triethylene glycoloxy linker while the mitomycin C derivative (porfirimycin) incorporated a complementary 7-N-terminal alkyne. The two components were ligated using the Huisgen [3 + 2] cycloaddition ("click") reaction. Preliminary biological assays demonstrated that the final hybrid compound retained both potent anti-estrogenic and anti-proliferative activities.

Aflatoxin B(1) (AFB(1) ) is a potent mutagen and an important risk factor for hepatocellular carcinoma (HCC) in humans. Transgenic mouse strains and cells in culture have been used to detect different types of mutations caused by AFB(1) and investigate the molecular determinants of their location and frequency. The AFB(1) mutational spectrum in the gpt gene was markedly different in AS52 cells compared with the liver in gpt delta B6C3F1 transgenic mice. The results demonstrate the importance of metabolism, chromosomal location, transcription and selection conditions on mutational spectra., (Copyright © 2012 Wiley Periodicals, Inc.)

Aflatoxin B (1) (AFB(1)) is a risk factor for hepatocellular carcinoma in humans. Infant, but not adult, mice are sensitive to AFB(1)-induced liver carcinogenesis; a single dose during the neonatal period leads to hepatocellular carcinoma in adulthood. Earlier work defined the mutational spectrum in the gpt gene of gpt delta B6C3F1 mice 3 weeks after exposure to aflatoxin. In the present study, we examined the gpt spectrum 10 weeks postdosing and expanded the study to examine, at 3 and 10 weeks, the spectrum at a second locus, the red/gam genes of the mouse λEG10 transgene. Whereas the gpt locus is typically used to define local base changes, the red/gam genes, via the Spi(-) assay, often are used to detect more global mutations such as large deletions and rearrangements. Three weeks after dosing with AFB(1), there was a 10-fold increase over the control in the Spi(-) mutant fraction (MF) in liver DNA; after 10 weeks, a further increase was observed. The MF in the gpt gene was also increased at 10 weeks compared with the MF at 3 weeks. No gender-specific differences were found in the Spi(-) or gpt MFs. Whereas Spi(-) mutations often signal large genetic changes, they did not in this specific case. The Spi(-) spectrum was dominated by GC to TA transversions, with one exceptionally strong hotspot at position 314. Using two genetic loci, the data show a strong preference for the induction of GC to TA mutations in mice, which is the dominant mutation seen in people exposed to aflatoxin.

The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death.

Exposure to genotoxic chemicals at a young age increases cancer incidence later in life. Aflatoxin B(1) (AFB(1)) is a potent genotoxin that induces hepatocellular carcinoma (HCC) in many animal species and in humans. Whereas adult mice are insensitive to aflatoxin-induced carcinogenesis, mice treated with AFB(1) shortly after birth develop a high incidence of HCC in adulthood. Furthermore, the incidence of HCC in adult male mice treated as infants is much greater than in females, reasons for which are unclear. In this study, treatment with AFB(1) produced similar levels of DNA damage and mutations in the liver of newborn male and female gpt delta B6C3F1 mice. Twenty-four hours after dosing with AFB(1) (6 mg/kg), the highly mutagenic AFB(1)-FAPY adduct was present at twice the level of AFB(1)-N(7)-guanine in liver DNA of males and females. A multiple dose regimen (3 × 2 mg/kg), while delivering the same total dose, resulted in lower AFB(1) adduct levels. Mutation frequencies in the gpt transgene in liver were increased by 20- to 30-fold. The most prominent mutations in AFB(1)-treated mice were G:C to T:A transversions and G:C to A:T transitions. At this 21-day time point, no significant differences were found in mutation frequency or types of mutations between males and females. These results show that infant male and female B6C3F1 mice experience similar amounts of DNA damage and mutation from AFB(1) that may initiate the neoplastic process. The gender difference in the subsequent development of HCC highlights the importance of elucidating additional factors that modulate HCC development.

Aflatoxin B₁ (AFB₁) is a DNA-binding toxin that contributes to the burden of liver cancer in tropical areas. AFB₁-DNA adducts are powerful biomarkers that discern individual and population risk from exposure to this carcinogen. The discovery of concordance between the metabolic pathways of the male Fischer rat and humans allowed data from rats to guide the development of chemoprevention strategies employed in clinical trials in high-risk regions. In this study, the variables of strain and sex are studied in the rat model, as a step toward understanding how ethnic differences and sex influence DNA adduct formation and the induction of enzymes by chemoprotective agents. Sulforaphane (SF), which induces phase II enzymes including glutathione S-transferases (GSTs), was evaluated for its ability to induce GST activity and reduce the AFB₁-DNA adducts in livers of both sexes of two rat strains that differ in susceptibility to AFB₁ hepatocarcinogenesis. A dose-dependent relationship was found for SF for both induction of GST and reduction in of AFB₁-N⁷-guanine in both Fischer (sensitive to AFB₁) and Sprague-Dawley rats (relatively resistant). Sprague-Dawley rats exhibited the greatest increase in GST levels and the largest reduction in AFB₁-N⁷-guanine in liver DNA. Males and females of each strain were also compared to determine if the ability of SF to induce GST and reduce AFB₁-N⁷-guanine correlated with gender differences in sensitivity to AFB₁ carcinogenesis. No gender-specific responses to SF were observed. These results support the view that SF induction of liver GST activity may play a role in its chemoprotective activity.

A strategy is described for the re-design of DNA damaging platinum(II) complexes to afford elevated toxicity towards cancer cells expressing the estrogen receptor (ER). Two platinum-based toxicants are described in which a DNA damaging warhead, [Pt(en)Cl(2)] (en, ethylenediamine), is tethered to either of two functional groups. The first agent, [6-(2-amino-ethylamino)-hexyl]-carbamic acid 2-[6-(7alpha-estra-1,3,5,(10)-triene)-hexylamino]-ethyl ester platinum(II) dichloride ((Est-en)PtCl(2)), terminates in a ligand for the ER. The second agent is a control compound lacking the steroid; this compound, N-[6-(2-amino-ethylamino)-hexyl]-benzamide platinum(II) dichloride ((Bz-en)PtCl(2))), terminates in a benzamide moiety, which lacks affinity for the ER. Using a competitive binding assay, Est-en had 28% relative binding affinity (RBA) for the ER as compared to 17beta-estradiol. After covalent binding to a synthetic DNA duplex 16-mer, the compound retained its affinity for the ER; specificity of the binding event was demonstrated by the ability of free 17beta-estradiol as a competitor to disrupt the DNA adduct-ER complex. The (Est-en)PtCl(2) compound showed higher toxicity against the ER positive ovarian cancer cell line CAOV3 than did the control compound. (Est-en)PtCl(2) was also more toxic to the ER positive breast cancer line, MCF-7, than to an ER negative line, MDA-MB231.

The multifunctional molecule 11beta-dichloro consists of a ligand for the androgen receptor linked to a bifunctional alkylating group, permitting it to create DNA adducts that bind the androgen receptor. We propose that binding of the androgen receptor to 11beta-DNA adducts acts to both shield damaged sites from repair and disrupt the expression of genes essential for growth and survival. We investigated the formation 11beta-DNA adducts in tumor xenograft and nontumor tissues in mice. Using [14C]-11beta-dichloro, we show that the molecule remains intact in blood and is widely distributed in mouse tissues after i.p. injection. Covalent 11beta-guanine adducts identified in DNA that had been allowed to react with 11beta-dichloro in vitro were also found in DNA isolated from cells in culture treated with 11beta-dichloro as well as in DNA isolated from liver and tumor tissues of mice treated with the compound. We used accelerator mass spectrometry to determine the levels of [14C]-11beta-DNA adducts in LNCaP cells treated in culture as well as in liver tissue and LNCaP xenograft tumors in treated mice. The level of DNA adducts in tumor tissue was found to be similar to that found in LNCaP cells in culture treated with 2.5 micromol/L 11beta-dichloro. Our results indicate that 11beta-dichloro has sufficient stability to enter the circulation, penetrate tissues, and form DNA adducts that are capable of binding the androgen receptor in target tissues in vivo. These data suggest the involvement of our novel mechanisms in the antitumor effects of 11beta-dichloro.

The goal of our work was the design of DNA-damaging agents that disrupt both DNA repair and signaling pathways in prostate tumor cells. A DNA alkylator (N,N-bis-2-chloroethyl aniline) was linked to a steroid ligand (17beta-hyroxy-estra-Delta(4(5),9(10))-3-one) to produce a complex molecule (11beta-dichloro) that forms DNA adducts that bind the androgen receptor (AR). We speculated that DNA adducts in an AR-DNA adduct complex would be camouflaged from DNA repair proteins that would otherwise remove the adducts in prostate cancer cells. Furthermore, transcription dependent on the AR would be antagonized by AR redistribution to sites distant from AR-driven promoters. The anticancer potential of 11beta-dichloro was demonstrated against prostate cancer cells in vitro and in vivo. 11beta-dichloro induces a unique pattern of gene disruption, induces apoptosis in apoptosis-resistant cells, and shows promising anticancer activity in animals.

A series of bifunctional compounds was prepared consisting of 17beta estradiol linked to a DNA damaging N,N-bis-(2-chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with DNA and binding of the resultant covalent adducts to the estrogen receptor. Linker characteristics were pivotal determinants underlying the ability of the compounds to kill selectively breast cancer cells that express the estrogen receptor.

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.

It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4'-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(-) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard-phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard-DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.

There is much interest in incorporating knowledge of biological mechanisms of carcinogenesis into assessments of health risks to humans posed by chemicals in the environment. Debate over the soundness of using data from animal bioassays conducted at minimally toxic doses or fractions thereof for predicting cancer risks to humans exposed to much lower doses has stimulated interest in the question of whether genotoxic or mitotic effects predominate in chemical carcinogenesis. Cell division plays a key role at each stage in the evolution of cancer, and it is well documented that increased rates of cell proliferation can escalate the risk of malignancy. This article examines the current understanding of both mechanisms by which chemicals provoke cell proliferation and the contribution of various kinetic patterns of cell proliferation to carcinogenesis.

High-pressure liquid chromatography can separate eight metabolites of benzo[a] pyrene formed by rat liver microsomes. This method offers major advantages over previous techniques used for the separation of oxygenated polycyclic aromatic hydrocarbons.

Normal cells cannot initiate DNA synthesis under inadequate external conditions, yet after growth has started they complete their division cycle under these conditions. The sensitive biochemical event for a growing cell is proposed to be accumulation of a labile protein which in adequate amounts permits entry into S phase, after about 2 hr, and completion of the cycle. Instability of this protein (half-life about 2.5 hr) creates a dynamic state so that its accumulation depends on rates of both synthesis and degradation. Neoplastic cells may show poorly regulated growth either by synthesizing this protein more rapidly or degrading it less rapidly, under conditions that limit normal cells' growth. Known mechanisms of overproduction include: more copies of the protein's structural gene per cell, an adjacent high-activity promoter, or autoproduction of growth factors. Less rapid degradation could result from less protease activity or from stabilizing modifications of the protein. Thus, derangement in the control of a labile growth-regulatory protein acting by any one of these diverse mechanisms could lead to neoplasia.

The formation of covalent aflatoxin-DNA adducts has been studied in embryo and yearling stages of the rainbow trout (Salmo gairdneri). A linear relationship was found between the amount of aflatoxin B1 (AFB1) absorbed into 21-day-old eggs and the level of covalent modification of embryo DNA after exposure to 0.25 to 0.5 p.p.m. aqueous solutions of AFB1 for 1 h; higher concentrations resulted in absorption of a greater proportion of AFB1. The principal covalent product, identified after acid and enzymatic hydrolysis of isolated embryo DNA, was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy aflatoxin B1. Additional AFB1 derivatives which were present are believed to be formed by chemical transformation of this product in DNA producing an aflatoxin-formamidopyrimidine adduct and the metabolic activation of other aflatoxin metabolites, such as aflatoxin M1 and aflatoxin P1. Although the eggs were exposed to AFB1 for a short time, covalent modification of DNA was evident over an extended period. The highest level of covalent products was present at approximately 24 h after exposure to 0.5 p.p.m. AFB1. Ninety-six hours later, this level had decreased slightly; however, the distribution of covalent adducts had changed: formamidopyrimidine adducts now represented up to 50% of the hydrolyzed aflatoxin derivatives. A similar pattern of covalent aflatoxin derivatives was found in the liver DNA of yearling trout 10 h after the administration of 0.3 mg/kg AFB1.

Primary cell cultures derived from human skin epithelium metabolized benzo(a)pyrene to three classes of compounds: phenols, quinones, and dihydrodiols. The relative proportions of metabolites varied according to the skin donor but differed from the pattern of metabolites in rat liver microsome preparations. While appreciable amounts of 7,8- and 9,10-dihydrodiol; 1,6-, 3,6-, and 6,12-quinone; and 3- and 9-hydroxy derivatives were found in the medium, no 4,5 (K-region)-dihydrodiol or epoxide was detected. Reduced amounts of quinones were produced when the cultures were pretreated with hydrocortisone before exposure to the hydrocarbon. The cultures did not require a period of enzyme induction for efficient metabolism of the hydrocarbon. Cultures of fibroblasts derived from the same skin samples as the epithelial cells metabolized the hydrocarbon but to a much different extent. Preexposure of the epithelial cell cultures to mixtures of polycyclic hydrocarbons resulted in a decrease in the amounts of carcinogen metabolized to phenols and dihydrodiols. These findings suggest that the prevalence of carcinomatous disease in humans is due to the differential capacity of the epithelial cells to metabolize potential carcinogens to active forms, a capacity reduced in fibroblasts or other nonepithelial cells. This suggestion is supported by the observations that supposedly normal prostate cells also efficiently metabolize polycyclic hydrocarbons in a manner similar to that of epidermal cells. No evidence of neoplastic transformation was seen in cytological preparations of cells exfoliated into the medium.

The products of in vivo covalent binding of activated aflatoxin B1 (AFB1) to DNA have been investigated in rats. The principal covalent product formed in liver DNA of rats treated with AFB1 has been identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1. This compound was isolated from the liver DNA of rats dosed with AFB1 (2.0 mg/kg) in sufficient quantity for characterization by physicochemical techniques, including field-desorption mass spectrometry. This information together with results of chemical methylation of the compound proved that the major adduct formed between DNA and AFB1 in vivo is identical to that produced in vitro when AFB1 is incubated with DNA in the presence of a rat liver microsomal activating system. Quantitative studies of formation of this compound revealed a dose-dependent relationship between the level of its occurence in liver DNA and AFB1 doses over the range 0.125-1.0 mg/kg.

The separation of ten isomeric benzo(a)pyrene phenols has been accomplished by the use of high-pressure liquid chromatography utilizing a newly developed recycling technique and new column and solvent systems. Using this new system and comparing the metabolites obtained with authentic standards, we have isolated 1-hydroxybenzo(a)pyrene and 7-hydroxybenzo(a)pyrene and identified them as metabolites formed by rat liver microsomes. In previously reported chromatography systems, the new metabolites migrated with another metabolite, 3-hydroxybenzo(a)pyrene.

Differences in benzo(a)pyrene metabolite pattern have been shown by rodent liver microsomes (Sprague-Dawley) and rodent embryo cells from Syrian hamsters and NIH Swiss mice. Rodent liver induced by methylcholanthrene shows marked quantitative variation between species. Additional pattern changes were found in mouse and hamster embryo secondary cultures with a reduction of the K-region metabolites and a marked increase in 9-hydroxybenzo(a)-pyrene. These results are indicative of a region-specific attack on the carcinogen by the cell monooxygenases which is distinct from the liver attack of microsomal enzymes on benzo(a)pyrene. These results suggest that activation and detoxification of benzo(a)pyrene may be species and tissue variable, and susceptibility and resistence to malignant transformation may be predicted on induction of a fortuitous combination of intermediate metabolic steps.

The covalent binding of the hepatocarcinogen aflatoxin B1 by rat liver microsomes to calf thymus DNA resulted in a binding level equal to one aflatoxin residue per 60 DNA nucleotides. An aflatoxin derivative-guanine adduct was efficiently liberated from DNA with formic acid. Analytical reversed-phase high-pressure liquid chromatography of the DNA hydrolysate revealed that approximately 90% of the carcinogen bound to DNA could be accounted for as a single component. Preparative high-pressure liquid chromatography was used to isolate sufficient quantities of the adduct for structural analysis from large quantities (340 mg) of DNA. A combination of spectral and chemical data indicates that the major product of the interaction of metabolically activated aflatoxin B1 and DNA is 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 with the guanine and hydroxyl functions possessing a trans configuration. The structural data support the hypothesis that the putative 2,3-oxide of aflatoxin B1 is quantitatively important as an intermediate in the binding of aflatoxin B1 to nucleic acids.

The chemical stability of aflatoxin B1 bound to calf thymus DNA was studied over a 48-hour exposure to phosphate buffers at pH 6.8-8.0 (37 degrees C). During this time, aliquots of the aflatoxin B1-modified DNA were acid-hydrolyzed and analyzed for the presence of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyflatoxin B1, 2,3-dihydro-2,3-dihydroxy-aflatoxin B1, and the tentatively identified, 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl-3-hydroxyflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1. Initial experiments determined the stability of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 in DNA at levels of modification of one residue per 60 and 1500 nucleotides. The acid-hydrolysis products obtained from these modified nucleic acids were qualitatively similar, but their proportional concentrations were different. These quantitative differences were dependent upon both pH and the initial level of modification of the DNA. During the first 6 hr of incubation, under all conditions examined, the formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B1 was responsible for the initial decrease of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct in DNA. After 48 hr of incubation at pH 7.0, the major reaction of the modified DNA was depurination of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct. However, at pH 8.0, the predominant reaction product formed in 48 hr was the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1. The putative DNA-bound products resulting from the elimination of the positive charge in the imidazole ring of the aflatoxin-N7-guanine adduct [2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1] were found to be stable in DNA for at least 24 hr at both pH 6.8 and 7.4. Taken together with observed patterns of stability of aflatoxin B1 adducts in vivo, these observations strongly suggest the involvement of enzymatic repair processes in removal of the N7-guanyl adduct and also emphasize the possible biological significance of the stable imidazole ring-opened adduct.

The metabolism of benzo(a)pyrene by human liver microsomes and human lymphocytes has been analyzed by high-pressure liquid chromatography. Human liver forms seven known metabolites and at least five additional unidentified metabolites that migrate as distinct peaks. Lymphocytes incubated with benzo(a)pyrene for 30 min do not form dihydrodiols. Lymphocytes incubated for 24 hr with benzo(a)pyrene form all of the metabolites produced by liver including dihydrodiols as well as additional metabolites. The ratios of phenols formed by liver and lymphocytes are different, and preparations from humans form a different profile of metabolites than that formed by rat liver.

Aflatoxin B1 and benzo(a)pyrene were activated by both cultured human bronchus and human colon as measured by binding to cellular DNA and protein. The binding of aflatoxin B1 to DNA was dose dependent, and the level of binding was higher in cultured human bronchus than it was in the colon. When compared to aflatoxin B1, the binding level of benzo(a)pyrene to both bronchial and colonic DNA was generally higher. The major adducts formed in both tissues by the interaction of aflatoxin B1 and DNA were chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (Structure 1) with the guanyl group and hydroxy group in trans-position and an adduct which has been tentatively identified by other investigators as 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (Structure 11). Seventy % of the radioactivity associated with bronchial DNA was found in these two peaks, and the ratio of radioactivity between the peaks was nearly 1. In colonic DNA, the ratio between Structures 1 and 11 was approximately 2. These observations add aflatoxin B1 to the list of chemical procarcinogens metabolized by cultured human tissues and in which the carcinogen-DNA adducts are similar to the adducts formed in animal tissue susceptible to the carcinogenic action of aflatoxin B1.

The covalent interactions between aflatoxin B1 (AFB1) and DNA were investigated in the inbred F344 rat and noninbred CD -1 Swiss mouse. A good correlation was found between the level of covalent modification of DNA, species sensitivity, and organ specificity, to the toxic effects of AFB1. The patterns of AFB1 acid hydrolysis products from DNA isolated from the livers and kidneys of both species were examined. The principal acid hydrolysis product in all cases was identified as 2,3-dihydro-3-hydroxy-(N7-guanyl)AFB1. Minor products consisted of adducts formed by the chemical transformation of the AFB1-N7-substituted guanine moiety forming putative formamidopyrimidine derivatives and the activation of AFB1 metabolites, which also modified the N-7 guanine atom. These last-mentioned products were present in greater amounts in resistant tissues. In vitro studies on the activation of AFB1 by microsomal fractions of mouse and rat livers found that mouse liver microsomes were rapidly inactivated.

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.