NAT1 is a critical prognostic biomarker and inhibits proliferation of colorectal cancer through modulation of PI3K/Akt/mTOR.

The aim of this study was to analyze the correlations between NAT1 and clinicopathological features of and prognosis in colorectal cancer (CRC). RNA sequencing data and clinical information were retrieved from The Cancer Genome Atlas database. Wilcoxon test, logistic regression and Kaplan-Meier method were used to estimate the association between NAT1 and prognosis in CRC. In vitro experiments were conducted to confirm the role of NAT1. NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. The authors further confirmed that expression of NAT1 was significantly lower in SW116 colon cancer cells than in NCM460 cells. Overexpressed NAT1 obviously inhibited the growth of CRC cells by downregulating phosphorylation of the PI3K/Akt/mTOR signaling pathway. NAT1 may be a potential therapeutic target for CRC.

Lay abstract Colorectal cancer (CRC) is a common malignancy worldwide. Because of the limited understanding of the pathogenesis and prognostic factors associated with CRC, the treatment effect in CRC remains poor. In the present study, the authors demonstrate that NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. NAT1 may exert antitumor activity by inhibiting phosphorylation of the PI3K/Akt/mTOR signaling pathway. These results suggest that NAT1 may be a prognostic factor in and therapeutic target for CRC.

Small molecule colorimetric probes for specific detection of human arylamine N-acetyltransferase 1, a potential breast cancer biomarker.

The identification, synthesis, and evaluation of a series of naphthoquinone derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 are described. The compounds undergo a distinctive color change (red --> blue) upon binding to these human and mouse NAT isoenzymes driven by a proton transfer event. No color change is observed in the presence of functionally distinct but highly similar isoenzymes which are >70% identical. These molecules may be used as sensors to detect the presence of human NAT1 in cell lysates.

N-acetyltransferase ARD1-NAT1 regulates neuronal dendritic development.

ARD1 and NAT1 constitute an N-acetyltransferase complex where ARD1 holds the enzymatic activity of the complex. The ARD1-NAT1 complex mediates N-terminal acetylation of nascent polypeptides that emerge from ribosomes after translation. ARD1 may also acetylate the internal lysine residues of proteins. Although ARD1 and NAT1 have been found in the brain, the physiological role and substrates of the ARD1-NAT1 complex in neurons remain unclear. Here we investigated role of N-acetyltransferase activity in the process of neuronal development. Expression of ARD1 and NAT1 increased during dendritic development, and both proteins colocalized with microtubules in dendrites. The ARD1-NAT1 complex displayed acetyltransferase activity against a purified microtubule fraction in vitro. Inhibition of the complex limited the dendritic extension of cultured neurons. These findings suggest that the ARD1-NAT1 complex has acetyltransferase activity against microtubules in dendrites. Regulation by acetyltransferase activity is a novel mechanism that is required for dendritic arborization during neuronal development.

An N-ethyl-N-nitrosourea-induced mutation in N-acetyltransferase 1 in mice.

Genetic variation in human N-acetyltransferases (NAT) has been implicated in susceptibility to aromatic amine and hydrazine carcinogens and therapeutic drugs. There are mouse models for variability of human NAT1; however mice with genetic differences in Nat1 (corresponding to human NAT2), have not been available. N-Ethyl-N-nitrosourea (ENU) mutagenesis was used to create genetic variation in Nat1. Among a number of mutations identified, a base-pair change substituting threonine for isoleucine at position 95 was recovered and studied. Molecular models suggested that this substitution would alter substrate binding. Analysis of hepatic Nat1 activity with the selective substrate isoniazid showed that there was a significant reduction in enzymatic activity in the homozygous mutants compared to the parental strain.

Acetylation pharmacogenetics. The slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver.

The biochemical basis underlying the genetic polymorphism of drug N-acetylation was investigated using a combination of in vivo and in vitro assays for arylamine N-acetyltransferase (NAT) activity and content in human liver. The acetylator phenotype of 26 surgical patients was determined using caffeine as an innocuous probe drug by measurement of the 5-acetyl-amino-6-formylamino-3-methyluracil to 1-methylxanthine molar ratio in urine. Liver wedge biopsies from these patients and livers from 24 organ donors were then used for measurement of N-acetyltransferase activity with the substrate sulfamethazine and for quantitation of immunoreactive N-acetyl-transferase protein. In vivo (caffeine metabolites in urine) and in vitro (sulfamethazine acetylation) measures of N-acetyl-transferase activity correlated very highly (r = 0.98). Moreover, in all subjects tested, slow acetylation both in vivo and in vitro was associated with a decrease in the quantity of immunodetectable N-acetyltransferase protein in liver cytosol relative to that seen in cytosols from rapid acetylator livers. Two kinetically distinct enzyme activities, designated NAT-1 and NAT-2, were partially purified from low- and high-activity livers and their relationship to acetylator status was determined. Low acetylation capacity was related to decreases in the liver content of both of these immunologically related proteins. The results demonstrate that genetically defective arylamine N-acetylation is due to a parallel decrease in the quantity of two structurally and functionally similar acetylating enzymes.

Microbial transformation of aniline derivatives: regioselective biotransformation and detoxification of 2-phenylenediamine by Bacillus cereus strain PDa-1.

A bacterial isolate, strain PDa-1, grew well on basal medium supplemented with 2-phenylenediamine, sucrose, and ammonium nitrate and completely transformed 2-phenylenediamine. The isolate was identified as Bacillus cereus. The product formed from 2-phenylenediamine was identified by EI-MS and NMR as 2-aminoacetanilide; whole cells converted 2-phenylenediamine to the product with a 76% molar yield. Whole cells also showed a broad substrate specificity toward 20 of 26 tested arylamines with substituent groups of various size and positions. Especially 2-aminobenzoic acid, 4-aminosalicylic acid, 5-aminosalicylic acid, and 2-aminofluorene were converted completely to the corresponding product with an aminoacetyl group. Cell extracts of strain PDa-1 had a high arylamine N-acetyltransferase activity. The partially purified enzyme converted 2-phenylenediamine to 2-aminoacetanilide. Strain PDa-1 constitutively expressed the enzyme in the absence of 2-phenylenediamine. Effects of 2-phenylenediamine and 2-aminoacetanilide on growth indicated that this enzyme probably plays a role in the detoxification of toxic arylamines in this strain.

Polymorphic expression of acetyl coenzyme A-dependent arylamine N-acetyltransferase and acetyl coenzyme A-dependent O-acetyltransferase-mediated activation of N-hydroxyarylamines by human bladder cytosol.

Human epidemiological studies suggest a genetic predisposition to bladder cancer among slow N-acetylators. The capacity of human bladder to N-acetylate arylamines, catalyzed by acetyl coenzyme A-dependent N-acetyltransferase(s) (EC 2.3.1.5) (NAT), may be an important step in the activation and/or deactivation of arylamines in the pathways leading to the initiation of bladder cancer. Another possible activation step is the direct O-acetylation of N-hydroxyarylamines via O-acetyltransferase(s) (OAT) to DNA-binding electrophiles. Human bladder cytosol from nine fresh autopsy specimens were investigated for NAT activity towards p-aminobenzoic acid, and the arylamine carcinogens 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Apparent Km determinations indicated little difference in NAT affinity (100-300 microM) for any of the substrates between the nine individual bladders. However, the apparent Vmax determinations indicated that the bladders could be classified into rapid or slow acetylator phenotypes based on their NAT activity towards 4-aminobiphenyl, 2-aminofluorene, and beta-naphthylamine. Four of the bladder cytosols had mean activities significantly (P less than 0.01) higher (approximately 10-fold) than the mean NAT activities of the other five bladder cytosols towards each arylamine carcinogen. However, no significant difference was detected in their NAT activities using p-aminobenzoic acid as a substrate. The human bladder cytosols were also tested for their capacity to activate N-hydroxy-3,2'-dimethyl-4-aminobiphenyl to a DNA-binding electrophile through a direct OAT-mediated catalysis. The N-hydroxyarylamine OAT activity also discriminated between two levels of activation, being significantly (P = 0.0002) higher (about twofold) in the rapid N-acetylator bladder cytosols, that correlated (r = 0.94) with the measured levels of NAT activity in each bladder cytosol. These results suggest that NAT activity and OAT activity of the human bladder vary concordantly with N-acetylator phenotype. The polymorphic expression of these acetylation activities may be important risk factors in human susceptibility to bladder cancer from arylamine carcinogens.

Assay for acetyl-CoA:arylamine N-acetyltransferase by high-performance liquid chromatography applied to serotonin N-acetylation.

A specific assay to measure the activity of the enzyme acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver is described. The assay is based on the HPLC analysis of N-acetylserotonin formed by the enzymatic reaction. A reversed-phase column (Spherisorb 5-microns ODS 2; 150 x 3.2 mm) eluted with 0.1 M sodium acetate (pH 4.75)/methanol (75:25) permits baseline separation of serotonin and N-acetylserotonin within 5.3 min. Several variables on the enzyme reaction were studied to obtain maximum activity. The enzyme is most active in glycine buffer at pH 9.5. The apparent Km value for serotonin (at 0.6 mM CoASAc) is 0.246 mM and 9.9 microM for CoASAc (at 1.5 mM serotonin). To avoid acetyl-CoA or N-acetylserotonin consumption in side-reactions, the enzyme was purified. A two-step purification process (ammonium sulfate fractionation and affinity chromatography on immobilised amethopterin) yielded 60-70% of the initial enzyme activity with a purification factor of 455-560.

Effects of 5-methoxypsoralen (5-MOP) on arylamine N-acetyltransferase activity in the stomach and colon of rats and human stomach and colon tumor cell lines.

BACKGROUND: It has been shown that cytochrome P450 enzymes (CYPs) and acetyltransferase can be used as biomarkers of carcinogen-DNA adduct levels and human cancer susceptibility. The gastrointestinal tract is the portal of entry of foreign compounds and presents xenobiotic metabolizing N-acetyltransferase (NAT) and CYPs activities. 5-Methoxypsoralen (5-MOP) has been used in combination with UV radiation in skin photochemotherapy for decades. A number of studies have demonstrated that 5-MOP is inhibitory towards mouse and human CYP isoforms, but investigations on the direct effects on NAT activity in laboratory animals and human cancer cells are limited. The main objective of this study was to document the effects of 5-MOP on the modulation of NAT activities in the stomach and colon of rats and human stomach and colon tumor cell lines. MATERIALS AND METHODS: N-Acetylation of 2-aminofluorene (AF) to 2-acetylaminofluorene (AAF) by NAT in the stomach and colon of Sprague-Dawley (SD) rats and in human stomach (SC-M1) and colon (COLO 205) tumor cell lines was investigated. RESULTS: The data show that the metabolic activity of NAT in the rat colon was higher than that in the rat stomach, and the further metabolism of AAF was slower in the stomach than in the colon. 5-MOP increased the activity of NATand also increased the further metabolism of AAF at 24 h in the rat stomach. In the rat colon, no statistically significant changes caused by 5-MOP were observed in NAT activity, but 5-MOP increased the further metabolism of AAF at 24 to 72 h. 5-MOP decreased the activity of NAT only at 72-h incubation in SC-M1 cells. In COLO 205 cells, however, 5-MOP decreased the activity of NAT between 24 h and 72 h. The optimal concentrations of 5-MOP to induce decreased NAT activity in SC-M1 cells were 0.05 mM to 25 mM. In COLO 205 cells, the data indicate that the higher the concentrations of 5-MOP, the higher the acetylation of AF; a promotion effect of NAT activity occured at a higher dose (50 mM) of 5-MOP and an inhibition effect occured at lower doses (0.05-0.5 mM) of 5-MOP, while concentrations of 5-25 mM of 5-MOP showed no significant difference compared with the control regimen. CONCLUSION: The metabolic activity of NAT in the rat colon was higher than that in the rat stomach, and the results also showed a high degree of correspondence with SC-M1 cells and COLO 205 cells. 5-MOP more efficiently inhibited NAT activity in human stomach and colon tumor cell lines than in the stomach and colon of rats.

Identification and characterization of two isozymic forms of arylamine N-acetyltransferase in Syrian hamster skin.

Arylamine N-acetyltransferase (EC 2.3.1.5) activity was examined using skin from Syrian hamster. Two isozymes of arylamine N-acetyltransferase, designated NAT-1 and NAT-2, were detected on anion-exchange high-performance liquid chromatography analysis. Both enzyme activities had indistinguishable molecular masses (30 kDa), but differed significantly in their specificity toward the aromatic amines including serotonin, dopamine, methoxytryptamine, tryptamine, para-phenetidine, para-aminobenzoic acid, and sulphamethazine. Specifically, NAT-2 but not NAT-1 catalyzed acetylation of dopamine to N-acetyldopamine and acetylation of serotonin to form N-acetylserotonin, a direct precursor of melatonin. The two isozymes were also distinguishable based upon their sensitivity toward methotrexate inhibition (50% inhibiting dose for NAT-1 = 380 microM; NAT-2 > 2 mM). The presence of these two activities in the skin raises new questions about the physiologic role of this enzyme in general and in the skin-specific functions in particular.

Chronic fluoxetine administration increases the serotonin N-acetyltransferase messenger RNA content in rat hippocampus.

BACKGROUND: It has been proposed that up-regulation of cyclic adenosine monophosphate response element binding protein is a common action of chronic antidepressant treatments that may regulate specific target genes in the hippocampus. We hypothesized that the serotonin N-acetyltransferase (AA-NAT; EC 2.3.1.87) gene is one such target. AA-NAT leads to formation of N-acetylserotonin from serotonin, and in the pineal gland, to melatonin synthesis. We investigated whether hippocampal AA-NAT expression can be modified by chronic administration of fluoxetine to rats. METHODS: Male Brown-Norway rats were administered 5 mg/kg fluoxetine or its vehicle either once (acute) or once daily for 21 days (chronic). They were sacrificed 18 hours after the last injection, and their hippocampi were processed for a quantitative reverse-transcription/polymerase-chain reaction assay of AA-NAT and cyclophilin (cyc) messenger (m)RNAs. The results are expressed as AA-NAT/cyc ratios. RESULTS: Chronic but not acute fluoxetine administration resulted in about a fivefold increase in hippocampal AA-NAT mRNA. CONCLUSIONS: Up-regulation of extrapineal, e.g., hippocampal, AA-NAT expression may play a role in mediating the therapeutic action of antidepressant drugs.

Is absence of pyruvate dehydrogenase complex in mitochondria a possible explanation of significant aerobic glycolysis by normal human leukocytes?

The oxygen consumption of leukocyte mitochondria of both healthy donors and leukemic patients was tested by using different respiratory substrates. The results indicate that pyruvate could not be utilized by mitochondria of normal leukocytes, whereas mitochondria of leukemic leukocytes could use pyruvate as a good respiratory substrate. A search for the possible presence of pyruvate dehydrogenase complex (PDC) in leukocytes indicates that this enzyme is apparently absent in mitochondria of normal leukocytes but is quite active in mitochondria of leukemic leukocytes. The absence of PDC in normal leukocyte mitochondria can explain the phenomenon of significant aerobic glycolysis that has been observed in normal leukocytes.

Exposure levels and cytochrome P450 1A2 activity, but not N-acetyltransferase, glutathione S-transferase (GST) M1 and T1, influence urinary mutagen excretion in smokers.

We investigated the polymorphic enzymes cytochrome P450 1A2 (CYP1A2), N-acetyltransferase (NAT2), glutathione S-transferase (GST) M1 (GSTM1), and T1 (GSTT1) in relation to cigarette smoking-associated urinary mutagenicity detected on YG1024 Salmonella typhimurium strain with S9 mix in 97 smokers. In each subject, cigarette smoke intake was checked by analysis of urinary nicotine plus its metabolites. NAT2 and CYP1A2 phenotypes were determined by the molar ratio of urinary caffeine metabolites detected by high-performance liquid chromatography, and GSTT1 and GSTM1 genotypes were determined by PCR. An increase in urinary mutagenicity was significantly related to levels of exposure to cigarette smoke and CYP1A2 N-hydroxylation activity (linear multiple regression analysis t = 4.51 and P < 0.001 and t = 3.09 and P = 0.003; F = 6.31, P < 0.001). Urinary mutagenicity was significantly higher in CYP1A2 extensive metabolizer smokers (n = 49) than in CYP1A2 poor metabolizer ones (n = 48; 2176 +/- 1525 versus 1384 +/- 1206 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.65, P < 0.001). The highest mutagenic activity was seen in subjects CYP1A2 extensive metabolizer/NAT2 slow acetylators (n = 29) with respect to the other phenotype combinations (n = 68; 2392 +/- 1660 versus 1525 +/- 1238 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.37, P = 0.017). NAT2 acetylation activity was slightly but inversely related to urinary mutagenicity, and the association was not significant. No effect of GSTM1 and GSTT1 genotypes in lowering (detoxifying) urinary mutagens was found. The significant enhancement of urinary mutagenicity associated with increased CYP1A2 activity, as already seen for diet-caused urinary mutagenicity, allows for many analogies between the process of mutagen formation derived from cooked meat and that from cigarette smoke condensate. In conclusion, the intensity of tobacco smoke exposure, modulated by CYP1A2 activity, is the major determinant of mutagenic urine among smokers, whereas GSTM1 and GSTT1 genotypes have no influence on this biomarker. This study suggests that CYP1A2 should definitely be determined in future studies involving urinary mutagenicity in cases in which smoking is a factor.

Alterations in the activities of tyrosinase, N-acetyltransferase, and tyrosine aminotransferase in immune reactive larvae of Drosophila melanogaster.

The activities of three enzymes, tyrosinase (monophenol oxidase, MPO), N-acetyltransferase (NAT), and tyrosine aminotransferase (TAT), were studied during eumelanotic encapsulation in host larvae of Drosophila melanogaster parasitized by the wasp, Leptopilina boulardi. At 24 h postinfection there was a tenfold increase in the MPO, whereas the activities of NAT and TAT were lower than those of nonparasitized controls. The data suggest that certain developmental processes are temporarily interrupted and alterations made in the metabolism of tyrosine to provide the metabolites necessary for a successful immune response. Two strains of D. melanogaster, R and Tyr-1, were parasitized and found to be immune reactive. The Tyr-1 strain is deficient in tyrosinase during the adult stage, but this mutation was found not to affect the immune capacity of the larvae. This is the first study to document concurrent alterations in the activities of various catecholamine-metabolizing enzymes during an immune response in an insect.

Evidence for the lack of hepatic N-acetyltransferase in suncus (Suncus murinus).

The abilities of liver cytosol fractions from the suncus and Sprague-Dawley (SD) rats to N-acetylate aniline, p-aminobenzoic acid, p-aminosalicylic acid and 2-aminofluorene (AF) were compared. The cytosol from rats N-acetylated these substrates at efficient rates, whereas the cytosol from the suncus did not N-acetylate these substrates at detectable rates. When AF was given to the suncus, 2-acetylaminofluorene (AAF), a metabolite of AF formed by N-acetyltransferase (NAT), was not detectable in serum, whereas the metabolite was seen clearly in rats. Northern blot and Southern blot analyses, using cDNAs coding for human NATs as probes, indicated that not only the transcripts but also the genes of the enzymes were undetectable in suncus. These results suggest that the suncus is among the few species known to lack NATs.

Cellular distribution of N-acetyltransferase activity in the rat small intestine.

The cellular distribution of AcCoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5) activities was examined in the rat small intestine to determine if heterogeneous cellular distribution contributes to preferential tumor development in the colonic region after exposure to heterocyclic amines (HAs). A chelation/elution method was used to preferentially isolate villus-tip, mid-villus, and crypt enterocytes. Monomorphic (NAT1) and polymorphic (NAT2) activities were determined using N-acetylprocainamide and N-acetamidobenzoic acid formation, respectively. Sucrase-isomaltase (SI) activity was used to confirm that a villus, mid-villus, and crypt cell gradient had been obtained. Utilizing this marker of villus enrichment, a 4- to 10-fold gradient was achieved. NAT1 and NAT2 activities followed this gradient, with the highest NAT activity occurring in the fraction with the highest SI activity. The ratio of NAT2:NAT1 remained essentially constant along the gradient, indicating a similar pattern of expression for both enzymes. This pattern of cellular distribution for the NATs is similar to that reported for cytochrome P450s. This apparent preferential expression of NAT in the villus cells may result in delivery of bioactivated HAs to the lower regions of the intestines as the villus-tip cells are extruded into the intestinal lumen and enter the fecal stream.

Rapid and simple measurement of serotonin N-acetyltransferase activity by liquid biphasic diffusion assay.

We report here a rapid, simple, and accurate method to assay for serotonin N-acetyltransferase (NAT) activity. This assay relies on the selective diffusion of radiolabeled acetyltryptamine into a water-immiscible scintillation fluid. Unlike organic solvent extraction, thin-layer chromatography, or high performance liquid chromatography, the separation of acetyltryptamine from acetyl CoA and tryptamine is not required in the method. Moreover, the limit of sensitivity is less than 4 pmol of N-acetyltryptamine formed per sample. Enhancement of NAT activity upon beta-adrenergic receptor stimulation in the rat pineal gland was clearly detected with this method. In addition, the NAT activity measurements obtained with this method agreed quantitatively in the pineal gland and other brain tissues with the conventional organic solvent extraction method. The results suggest that this liquid biphasic diffusion assay is applicable to the detection of NAT activity in tissues and cells.

Melatonin and serotonin N-acetyltransferase activity in developing eggs of the cricket Gryllus bimaculatus.

Melatonin (N-acetyl-5-methoxytryptamine) and serotonin N-acetyltransferase (NAT), a key regulatory enzyme in melatonin synthesis, are present in the adults and larvae of several insect species, as well as in vertebrates. To determine when melatonin and NAT first appear in insects ontogenetically, melatonin levels and NAT-like activity were measured in developing eggs of the cricket Gryllus bimaculatus. When the eggs were incubated under a 12-h light/12-h dark (LD) cycle at 24-26 degrees C, melatonin was detected in the egg extracts at all of the developmental stages examined. NAT-like activity was first found in the eggs 3 days after oviposition. From 5 to 11 days after oviposition, both NAT-like activity and melatonin levels showed significant day/night changes with the high levels occurring during the dark period of the LD cycle. By contrast, significant day/night changes were not detected in eggs just before hatching. To determine more detailed temporal changes, NAT-like activity was assayed in eggs 6 to 7 days after oviposition at 2- or 4-h intervals over a 48-h period. The activity in the eggs clearly exhibited a diurnal rhythm, peaking in the dark period of the LD cycle, and the rhythm persisted in constant darkness. These results suggest that the cricket egg (probably the embryo) synthesizes melatonin, and that its melatonin synthesis may fluctuate with a circadian rhythm. In addition, the results of the present study strongly suggest that a circadian clock controlling NAT activity functions in the cricket at the embryonic stage.

A differential role of CREB phosphorylation in cAMP-inducible gene expression in the rat pineal.

In the rat pineal gland cAMP mediates nocturnal induction of the enzyme arylalkylamine N-acetyltransferase (AA-NAT) as well as of transcription factors such as inducible cAMP early repressor (ICER), Fos-related antigen-2 (Fra-2) and JunB. Cyclic AMP stimulates the phosphorylation of the DNA binding protein cAMP response element binding protein (CREB). While cAMP-induced CREB phosphorylation appears to be a prerequisite for AA-NAT and ICER gene expression, it is not known whether CREB phosphorylation accounts for the full cAMP response of the two genes. Furthermore, the significance of CREB phosphorylation in cAMP-activated Fra-2 and JunB transcription is unknown. In the present in vitro study we used the serine/threonine protein phosphatase inhibitor okadaic acid (OA) to phosphorylate CREB without altering intrapineal cAMP concentration. It was observed that OA (10(-7) M) was less effective than dibutyryl cAMP (dbcAMP; 10(-3) M) in inducing AA-NAT mRNA and ICER mRNA, respectively. On the basis of this finding, it is concluded that CREB phosphorylation alone is apparently not sufficient for the full cAMP response of the two genes. By contrast, OA and dbcAMP equally stimulated the accumulation of the mRNAs of Fra-2 and JunB. Therefore cAMP may induce Fra-2 and JunB transcripts via CREB phosphorylation. Our observations suggest that CREB phosphorylation plays a critical role in diversification of cAMP-dependent gene induction in the rat pineal.

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells.

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.