Binding of dimethylaminoazobenzene metabolites to DNA and proteins. I. In vitor studies on a microsomal dependent system

Incubating dimethylaminoazobenzene [aniline ring (U)¹⁴ C] (DAB), purified thymus DNA and rat liver microsomes led to a covalent binding of radioactivity to DNA and proteins, which implied the presence of bound DAB metabolites. This binding required an enzymatic activation. We studied some characteristics of the enzymatic process, i.e. NADPH requirement, effect of pH, effect of various concentrations of microsomes and DNA, induction by 3-methylcholanthrene. This in vitro system is discussed in view of its validity for studying interactions between the carcinogenic azodyes and the cellular constituents.     

The covalent binding of metabolites of dimethylaminoazobenzene, β-naphthylamine and aniline to nucleic acids in vivo

• 1. Following injection of dimethylaminoazobenzene-³H labelled in the primed ring (150 mg/kg) into male albino rats and guinea-pigs maintained on a diet containing 10% protein, radioactivity was covalently bound to r-RNA, DNA, cytoplasmic protein and nuclear protein of liver and spleen.
• 2. For liver the maximum levels of binding (μM/g × 10²) were r-RNA 11.4, cytoplasmic protein 54, nuclear protein 39, DNA 1.4; for spleen: r-RNA 1.0, DNA 0.5, cytoplasmic protein 89, and nuclear protein 39; and for guinea-pig liver r-RNA 3.4, DNA 2.2, cytoplasmic protein 55, nuclear protein 28, indicating a much higher ratio of protein binding to r-RNA binding than in the rat (16 compared with 5). The difference in ratio was also large for the binding to two transplanted hepatomas in August and Hooded rats compared with albino rat liver (37 and 13).
• 3. For rat liver r-RNA and protein the decay of radioactivity paralleled the rate of turnover of r-RNA (as measured by orotic acid-³H incorporation) and of protein. The level of DNA-bound radioactivity remained constant for 290 hours.
• 4. The radioactivity associated with the nucleic acids and proteins remained bound after treatment with 0.3 N KOH which converts the nucleic acids to nucleotides, and with Crotalus adamanteus venom which converts them to purine and pyrimidine nucleosides. Treatment with N HCl for 1 hr at 100°C which forms purine bases and pyrimidine nucleotides and degrades proteins into peptides and amino acids, liberated the majority of the radioactivity so that it became extractable at various pH values.
• 5. The binding of β-naphthylamine-³H and aniline-³H hydrochloride to rat liver, spleen and kidney were also studied. Binding occurred to DNA, ribosomal RNA and protein in each case. For β-naphthylamine the levels of metabolite binding were similar to dimethylaminoazobenzene metabolite binding to guinea-pig liver components. For aniline hydrochloride, binding to ribosomal RNA and protein was of a low order.
• 6. The possible relevance of these findings to the problem of carcinogenesis is discussed.

     

Labeling in cells of the nasal cavity of hamsters treated with tritiated N-nitrosodiethylamine

Syrian golden hamsters were given a single dose of N-[³H]nitrosodiethylamine ([³H]DEN) by gavage. One hour later the animals were killed and their nasal cavities processed for high resolution light microscopic autoradiography for detection of bound radioactivity. The atrioturbinals, which are lined by squamous epithelium, were unlabeled. In the respiratory portion of the nose (nasal septum, naso- and maxilloturbinals) most bound radioactivity was concentrated in the mucous cells of the respiratory epithelium and the secretory cells of submucous glands). In the olfactory region (ecto and endoturbinals) most bound radioactivity was restricted to the secretory cells of submucous glands, while in the olfactory epithelium only the sustentacular cells were slightly labeled.     

In vivo and in vitro binding of carbon tetrachloride with nucleic acids and proteins in rat and mouse liver

¹⁴C-labelled carbon tetrachloride binds in vivo to DNA of mouse liver and to ^rRNA of rat liver if the animals have been pretreated with 3-methylcholanthrene. A noticeable amount of radioactivity is also observed in liver proteins. In vitro carbon tetrachloride is activated by microsomes and pH 5 enzymes of 3-methylcholanthrene-treated animals to a metabolite which can react with DNA and polynucleotides; this effect is more evident if subcellular fractions from mouse liver are used.     

Effects of131I-EGF on cultured human glioma cells

Malignant glioma cells often have more epidermal growth factor (EGF) receptors than normal cells and targeting of toxic substances to the receptor might therefore be an attractive therapeutical approach. Radiation effects were analysed on human glioma cells growing as monolayers after exposure to¹³¹I-EGF. Unspecific effects were analysed with¹³¹I-BSA or after presaturation with nonradioactive EGF. The radiation effects were compared to the effects obtained by external⁶⁰Co gamma irradiation. Administration of the highest radioactive concentrations, 0.2–0.5 MBq/ml in the culture medium, corresponded, after 20 min incubation, to a binding of about 1.0–2.5 dpm/cell. Such an exposure to¹³¹I decays gave effects on cell survival corresponding to about 2.5 Gy of external gamma irradiation. Somewhat less than half of this effect came from the specific bound radioactivity and the rest from nonbound radioactivity. When administrating lower concentrations of radioactivity both the binding and the radiation effects were smaller. The observations showed that it is possible to inactivate cell-proliferation of glioma cells with specific bound¹³¹I-EGF. The possibilities to fractionate the treatments and of binding also other toxic agents than¹³¹I to the EGF receptor are discussed.     

A qualitative gas chromatographic analysis of substituted 5,6-dihydroxyindoles from the urine of patients with melanoma

A qualitative gas chromatographic analysis of trimethylsilylated ethyl acetate extracts of melanotic urine revealed 5 indolic compounds, which have been identified as substituted 5,6-dihydroxyindoles. Ethyl acetate extracts of melanotic urines at pH 2.0 contained isomeric 5-hydroxy-6-methoxy and 6-hydroxy-5-methoxy-indolyl-2-carboxylic acids which were not separable under the conditions used. A careful hydrolysis of melanotic urine with a Helix pomatia preparation followed by extraction at pH 6.5 in a nitrogen atmosphere released 3 additional indolic compounds from their conjugated form. Using gas chromatographic-mass spectrometric analysis they were identified as 5-hydroxy-6-methoxy, 6-hydroxy-5-methoxyindole and 5,6-dihydroxyindole.     

Differential distribution and covalent binding of two labeled forms of methyl-CCNU in the Fischer 344 rat

Summary The present study compares the organ distribution and covalent binding of MeCCNU labeled either within the carbamylating ([cyclohexyl-1-¹⁴C]MeCCNU; Chx-¹⁴C-MeCCNU) or alkylating ([2-chloroethyl-1,2-¹⁴C]MeCCNU; Cle-¹⁴C-MeCCNU) region of the compound in an animal model shown to be suitable for studying the nephrotoxicity of the nitrosoureas. Extraction of tissue homogenates with organic solvents of increasing polarity, and subsequent analysis of these extracts by HPLC showed fat to accumulate the highest concentration of parent compound. Kidney accumulated the highest levels of the more polar ether- and methanol-extractable metabolites and/or degradation products of either cyclohexyl-derived or chloroethyl-derived ¹⁴C-MeCCNU. Striking differences were apparent in the accumulation, degradation and/or metabolism, and tissue distribution of covalently bound radioactivity for the chloroethyl and cyclohexyl moieties. For example, approximately twice as much cyclohexyl-derived ¹⁴C was bound covalently to protein of kidney than to protein of liver or lung. In contrast, approximately twice as much chloroethyl-derived ¹⁴C was bound to lung protein than to liver of kidney protein. No radioactivity was bound covalently to tissue DNA following Chx-¹⁴C-MeCCNU administration. On the other hand, at 4 h, chloroethyl-derived ¹⁴C was irreversibly bound to DNA in the relative amounts of kidney (5.0 nmol/mg), liver (2.7 nmol/mg), and lung (1.25 nmol/mg). These results demonstrate that MeCCNU metabolites and/or degradation products are preferentially accumulated in kidney, a primary target organ for MeCCNU toxicity. Moreover, kidney protein and DNA were subject to extensive carbamylation and alkylation reactions as measured by irreversibly bound cyclohexyl-derived and chloroethyl-derived ¹⁴C, respectively. These data suggest that the extent of irreversibly bound drug to tissue macromolecules may be a valid predictor of MeCCNU toxicity. However, the relative toxicological significance of either protein carbamylation or DNA alkylation in mediating MeCCNU-induced nephropathy is not yet understood.Abbreviations MeCCNU 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea - Chx-¹⁴C-MeCCNU [cyclohexyl-1-¹⁴C]-MeCCNU - Cle-¹⁴C-MeCCNU [2-chloroethyl-1,2-¹⁴C]-MeCCNU] - HPCL high performance liquid chromotography - PAH p-aminohippuric acid     

A quantitative determination of the covalent binding of a series of polycylic hydrocarbons to dna in mouse skin

The extents of reaction of seven ³H-labelled polycyclic hydrocarbons with DNA in mouse skin following their topical application have been studied. DNA isolated from the treated areas was hydrolysed enzymically to deoxyribonucleosides and the products chromatographed on Sephadex LH20 columns. The levels of binding of hydrocarbon to DNA were determined from the amounts of radioactivity eluted from Sephadex LH20 columns in those fractions containing hydrocarbon-DNA adducts. Tritium incorporation into normal deoxyribonucleosides was examined by rechromatography of material on AG50W-X4 columns and in some instances was found to account for a substantial proportion of the total radioactivity associated with DNA samples. Following treatment of mice with 1 (imol hydrocarbon/mouse, 7, 12-dimethylbenz(a)anthracene, the most potent carcinogen studied, became bound to DNA to the greatest extent (43 pmol/mg DNA), Benzo(a)pyrene and 3-methylcholanthrene, the next most active compounds, became bound to DNA to similar extents (27 pmol/mg DNA and 25 pmol/mg DNA), as did 7-methylbenz(a)anthracene (25 pmol/mg DNA) although it is a less active compound. Dibenz-(a,h) anthracene, another moderately active hydrocarbon, became bound to DNA to the extent of 15 pmol/mg DNA, and the very weakly active compounds, benz(a)anthracene (2 pmol/mg DNA) and dibenz(a,c) anthracene (10 pmol/mg DNA) became bound to DNA to the lowest extents. Dibenz(a,h))anthracene differed from the other hydrocarbons in showing a maximum level of binding 72 h after treatment, compared with 19–24 h for the other compounds.     

DNA binding of iproplatin and its divalent metabolitecis-dichloro-bis-isopropylamine platinum(II)

Summary The quadrivalent second-generation platinum complex iproplatin and an in vivo divalent metabolite of iproplatin,cis-dichloro-bis-isopropylamine platinum (CIP) were tested for binding to DNA in vitro. DNA binding was determined according to radioactivity measured using [¹⁴C]-iproplatin and [¹⁴C]-CIP and also by platinum content. Results indicate that (a) iproplatin shows negligible binding to DNA, (b) CIP binds to DNA in a time-dependent fashion, and (c) the isopropylamine ligand is intact when CIP is bound to DNA. Glutathione (GSH) inhibits the binding of CIP to DNA, possibly by inhibiting binding to DNA of the aquated form of CIP.     

Erroneous identification of APOBEC3-edited chromosomal DNA in cancer genomics

Background:

The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5′TpC context. These can be copied as G->A transitions in the 5′GpA context.

Methods:

DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA.

Results:

Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5′GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR.

Conclusions:

Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼10⁴–10⁵ base substitutions per cancer genome, or 0.003–0.03 kb⁻¹. By contrast, the 3DPCR error rate is of the order of 4–20 kb⁻¹ owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5′GpC represent artefacts.     

Cytotoxic compounds from Annonaceus species as DNA topoisomerase I poisons

BACKGROUND: In the search for cytotoxic natural products as DNA topoisomerase poisons, we have assessed six annonaceus compounds (the acetogenins annonacin and rolliniastatin-1, the styryl-lactones etharversin and altholactone and the alkaloids thaligrisine and cepharanone-B) for cytotoxic activity against three human cancer cell lines and then we evaluated these compounds as DNA topoisomerase poisons. MATERIALS AND METHODS: The cytotoxicity parameters were determined following protocols established by the National Cancer Institute (NCI) using the SRB assay. In the topoisomerase assay, the supercoiled DNA produces open circle forms that are stabilised in the presence of DNA topoisomerase poisons and can be detected after a denaturation step by proteinase K-SDS. RESULTS: The six compounds showed cytotoxic activity, with cepharanone B being the most cytotoxic one, even more than the antineoplastic agent etoposide on two cancer cell lines, although it is the only one that does not act as a DNA topoisomerase poison. CONCLUSION: These results could justify the traditional use of the studied annonaceus species and topoisomerase-mediated DNA damage might be a possible mechanism by which five of these compounds exert their cytotoxicity.     

Halomethyl-1,2,3-triazole derivatives: A new type of alkylating agent active in mouse transplantable tumors

Summary A number of halomethyl-1,2,3-triazole derivatives have been tested for their effect on ICR Swiss female mice bearing the Ehrlich carcinoma ascites (ECA) tumor. Two of these compounds, namely 4-bromomethyl-1-(2,3,4,6-tetra-0-acetyl--D-glucopyranosyl)-1,2,3-triazole and 4-iodomethyl-1-(2,3,4,6-tetra-0-acetyl--D-glucopyranosyl)-1,2,3-triazole, referred to here as compounds H and K, respectively, promoted a significant increase in the median survival time (145% and 195%) when injected IP for 9 consecutive days at doses of 75 mg/kg/injection and 100 mg/kg/injection, respectively. DNA synthesis, as measured by (methyl-³H)thymidine incorporation into trichloroacetic acid-precipitated material, was always inhibited by all the triazole derivatives to a greater extent than the incorporation of (5,6-³H)uridine, (5-³H)proline, and (6-³H)-glucose. Moreover, inhibition of DNA synthesis was complete and irreversible following exposure to selected triazole derivatives. Compounds H and K inhibited the incorporation of di(³H)methyl sulfate by ECA cells. In addition, compound K promoted the release of radioactivity associated with trichloroacetic acid-insoluble material coming from (8-³H)guanosine-prelabeled cells. This release of radioactivity did not occur when cells were prelabeled with (methyl-³H)thymidine. It is concluded from these results that these triazole derivatives act as alkylating agents.     

Formation of covalent deoxyribonucleic acid benzo[a]pyrene 4,5-epoxide adduct in mouse and rat skin

The covalently bound products of [³H]benzo[a]pyrene (BP) were determined in the DNA from the skin of mice and rats. The quality and the distribution of bound products was similar. The formation of products corresponding to 4,5-dihydro-4,5-epoxy benzo[a]pyrene (BPE) and to a further metabolite of 9-hydroxybenzo[a]pyrene bound to DNA were found in the 2 species at the 24 h end point.     

Doxorubicin inhibits human DNA topoisomerase I

Summary Purified human DNA topoisomerase I was assayed quantitatively by enzyme titrations with supercoiled pHC624 DNA in the presence of 0–2.0 m doxorubicin. Supercoiled and relaxed DNAs were resolved by agarose gel electrophoresis in the presence of ethidium bromide, and the pereentage of conversion of supercoiled DNA to relaxed DNA was quantified by scanning microdensitometry. The inhibition of DNA topoisomerase I activity was measured at varying concentrations of doxorubicin. Doxorubicin inhibited enzyme activity at an IC₅₀ value (the concentration required to inhibit 50% of the total activity) of 0.8 m. Similar inhibition was observed for daunomycin, a structurally related anthracycline antitumor drug. These results indicate that anthracyclines inhibit human DNA topoisomerase I activity at concentrations that cause DNA damage and cytotoxicity in vivo.This investigation was supported by grants from the Research Council of Rutgers University and the Charles and Johanna Busch Bequest     

Cellular metabolism of 5,6-dihydro-5-azacytidine and its incorporation into DNA and RNA of human lymphoid cells CEM/O and CEM/dCk(-)

Summary 5,6-Dihydro-5-azacytidine (DHAC) is a hydrolytically stable analog of 5-azacytidine (5-aza-C) that has antileukemic activity against experimental leukemias and, like 5-aza-C, causes DNA hypomethylation. We report the cellular metabolism of DHAC and its incorporation into nucleic acids in the CCRF/CEM/O and deoxycytidine kinase mutant CCRF/CEM/dCk(-) human lymphoid cell lines. The cells were incubated with their respective IC₅₀ concentrations for 24 h, then aliquot samples were removed at predetermined intervals and extracted for nucleotides. The acid-soluble extracts of the cells were assayed on HPLC for nucleotides of DHAC. The major anabolite of [³H]DHAC, [³H]DHACTP, peaked at 110.3±30.7 M in CEM/O and at 96.3±41.9 M in CEM/dCk(-) cells at 9 and 12 h, respectively. The intracellular concentrations of the deoxyribonucleoside triphosphate, [³H]DHAdCTP, peaked at 13.5±7.7 M at 4 h in CEM/O and at 80.8±13.8 M at 12 h, a 6-fold greater cellular concentration, in the dCk mutant cell line. The amount of DHAC anabolites incorporated into CEM/O nucleic acids reached a plateau in RNA at 552.6±7.8 pmol/10⁷ cells and in DNA at 64.55±10.0 pmol/10⁷ cells. In CEM/dCk(-) cells, DHAC anabolites reached a plateau in RNA and DNA at 4,256.3±631.0 and 395.5±145.4 pmol/10⁷ cells, respectively. Thus, with equitoxic treatments of DHAC, the incorporation of its analog anabolites into RNA and DNA was 8- and 6-fold greater in CEM/dCk(-) cells. DNA methylation levels were depressed equally despite a 6-fold greater incorporation of the analog in DNA in the CEM/dCk(-) cells indicating that hypomethylation may be saturated after DHAC treatment. The DNA methylation levels reached a nadir of 0.19% and 0.20% methyl-C (percentage of methylation) in the two cell lines at 6 and 12 h after the beginning of drug treatment and remained relatively constant for the duration of the 24-h treatment. A curve-linear relationship was obtained between the DNA methylation levels in both cell lines and the amounts of DHAC anabolite incorporated into DNA.Abbreviations used 5-aza-C 5-azacytidine - DHAC 5,6-dihydro-5-azacytidine - DHACTP 5,6-dihydro-5-azacytidine 5-triphosphate - DHAdCTP 5,6-dihydro-5-azacytidine 5-deoxy-triphosphate - dCk deoxycytidine kinase - PCA perchloric acid - SAX strong anion exchange - PBS phosphate-buffered saline Supported by research grant CA 38905 from the National Institute of Health, NCI, and the T. J. Martell Foundation for Leukemia and Cancer Research, The Neil Bogart Memorial Laboratories     

Tricycloquinazoline carcinogenesis: interaction of carcinogen with mouse skin proteins

Following topical application of tricycloquinazoline-¹⁴C (0.03 μM) to mice, radioactivity was covalently bound to soluble and particulate skin proteins. The maximum level of binding to soluble proteins (equivalent to 4.2×10^?6 μM TCQ/mg) was reached after 24 hours and that to particulate proteins (2.5×10^?6 μM TCQ/mg) after 48 hours. These binding levels are lower than those observed with carcinogenic and non-carcinogenic hydrocarbons studied under similar conditions. Treatment with doses of tricycloquinazoline greater than 0.03 μM did not produce increased levels of binding. The radioactivity associated with proteins was liberated following treatments (4 N KOH or 6 N HCl) which extensively degrade proteins. None of this radioactive material was identifiable as unchanged tricycloquinazoline. In the soluble proteins, radioactivity was bound almost exclusively to protein having the mobility of albumin whilst no significant radioactivity was associated with h proteins.     

Apical release of base-labile fatty acyl groups commensurate with stimulation of glycoprotein sialosyl Lewisa secretion in colorectal carcinoma cells

The rate of polarized secretion of a putative adhesion ligand, sialosyl Lewis^a (19–9), by SW1116 colorectal carcinoma cells is stimulated at least 20-fold after pre-incubation with, and the incorporation of, retinoic acid (RA). In order to investigate the possible involvement of fatty acylation in the export of the epitope, purified ligandsfrom carcinoma-cell membranes, membrane subfractions and media were analyzed during RA-induced secretion. Incorporation of radioactivity from (³H)palmitate into membrane subfractions and purified sialosyl Lewis^a antigenic molecular species of Mr > 150,000 (SiaLeams) was stimulated by RA treatment. Most of the intracellular lipid radioactivity which bound to solid-phase 19–9 antibody behaved chromato-graphically, either like ganglioside or like NH₂ OH-labile acyl groups, but most of the (³H) bound to SiaLeams of post-incubation media behaved like base-labile fatty acyl groups, or free fatty acid. Release of base-labile lipid radioactivity after 3 hr (associated with antigen) was almost exclusively into the apical media of membrane inserts. Gas-liquid chromatography/mass spec, analyses of purified Sialeams revealed the presence of palmitate (16:0), as well as stearate (18:0) and oleate (18:1) fatty acyl groups. Our results suggest that fatty acylation of SiaLeams may be co-ordinated with alterations in glycosylation and participate in directing these molecules to the apical surface. Lipid analyses were consistent with ganglioside chaperonage of SiaLeams to the apical surface, where N-fatty-acylated gangliosides remain for the most part integrated into the bilayer, but some oxyester or thioester bonds may be cleaved to permit release of SiaLeams to the apical medium. © 1995 Wiley-Liss Inc.     

DNA-damage in human cells treated with the closely related alkylating agents peptichemio,m-l-sarcolysin and melphalan

In vitro experiments were performed on human peripheral leukocytes to compare DNA repair synthesis induced by peptichemio (a multipeptide complex containing m-l-sarcolysin), m-l-sarcolysin and melphalan. The peptide bound m-l-sarcolysin gave rise to maximum DNA repair synthesis at a concentration interval between 1 × 10⁻⁵ and 3 × 10⁻⁵ M as opposed to a concentration interval of 3 × 10⁻⁴ to 1 + 10⁻³ M for melphalan. The peptide bound m-l-sarcolysin induced DNA repair synthesis at similar molar concentrations to m-l-sarcolysin alone. The difference between melphalan on one hand, and m-l-sarcolysin and peptichemio on the other hand, may reflect a more rapid uptake of the latter substances. Inhibition of ³H-uridine incorporation in human peripheral lymphocytes supports-this hypothesis. The frequency of sister chromatid exchanges in human fibroblasts was significantly higher after treatment with m-L-sarcolysin compared to melphalan, a finding which also indicates that these chemically closely related substances modify the cellular DNA differently. When DNA repair synthesis was induced by u.v.-light and methyl-methane-sulphonate (a monofunctional alkylating agent), no additional DNA repair synthesis was induced, if peptichemio, m-L-sarcolysin or melphalan was added. These results indicate that the repair of lesions induced by melphalan peptichemio, u.v.-light and methylmethane sulphonate is mediated by at least one common controlling enzyme.     

Binding of [14C]azaserine to DNA and protein in the rat and hamster

The binding of [¹⁴C]azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various times after treatment with [¹⁴C]azaserine. The specific activity of ¹⁴C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.     

Nitric oxide: its production in host-cell-infiltrated EMT6 spheroids and its role in tumour cell killing by flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

Summary Flavone-8-acetic acid (FAA) and its more dose-potent analogue 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), appear to exert their antitumour effects through vascular and other host-mediated mechanisms and are known to induce the synthesis of nitric oxide by murine macrophages. We investigated the role of nitric oxide in the cytotoxic effects of these drugs in host-cell-infiltrated spheroids. EMT6 murine mammary adenocarcinoma cells were grown in culture to produce multicellular spheroids in vitro spheroids), which were then inoculated i. p. into mice. After 6 days the spheroids were removed ex vivo spheroids). Exposure to FAA (890 m) and 5,6-MeXAA (80 m) in vitro for 20 h increased nitrite concentrations to 6.7 and 9.7 nmol/spheroid, respectively, as compared with 0.7 nmol/spheroid in the absence of drug. FAA and 5,6-MeXAA did not increase nitrite production in in vitro spheroids in cells obtained by peritoneal lavage. However, mixed cultures of in vitro spheroids and peritoneal cells treated with 5,6-MeXAA produced nitrite (2.5 nmol/spheroid), indicating that interactions between host cells and tumour cells were important for induction. The effets of these drugs on ex vivo spheroids were prevented by co-incubation withN ^G-monomethyl-l-arginine, indicating that nitrite originated from the oxidation ofl-arginine to nitric oxide. Cell sorting of disaggregated spheroids into EMT6 cells andMac-1-positive macrophage populations indicated that both of these cell populations could be induced to synthesise nitric oxide by subsequent incubation with 5,6-MeXAA. Incubation of ex vivo spheroids with FAA and 5,6-MeXAA decreased the clonogenicity of EMT6 cells, and this effect was wholly (FAA) or partially (5,6-MeXAA) reversed by the presence ofN ^G-monomethylarginine (250 m). FAA and 5,6-MeXAA may therefore exert some of their cytotoxic effects on tumour cells through the production of nitric oxide.This work was supported by grants from the New Zealand Lotteries Board, the Cancer Society of New Zealand and the Health Research Council of New Zealand