A new approach for assessing the dietary exposure to food additives.

By analyzing composite samples of brand name foods, a new method called the Dietary Exposure Assessment Method (DEAM), if found to be feasible, may provide useful estimates of daily intake of food additives for major portions of the U.S. population.

Selective mouse breeding for short ethanol sleep time has led to high levels of hepatic aromatic hydrocarbon (Ah) receptor.

Following a selective breeding program of heterogeneous mice for more than 30 generations, SS ("short sleep") and LS ("long sleep") lines have been developed on the basis of their sleep times when challenged with a single intraperitoneal dose of ethanol. The aromatic hydrocarbon responsiveness (Ah) locus encodes the Ah receptor, which regulates the induction of certain drug-metabolizing enzymes by polycyclic aromatic compounds such as 3-methylcholanthrene and tetrachlorodibenzo-p-dioxin. The C57BL/6 inbred mouse strain (B6; Ahb/Ahb) has a high-affinity Ah receptor, while the DBA/2 inbred mouse strain (D2; Ahd/Ahd) has a low-affinity Ah receptor. We show here that the SS inbred mouse line exhibits markedly elevated hepatic levels of the high-affinity Ah receptor, while the LS outbred mouse line contains the low-affinity Ah receptor. Among progeny of (B6D2)F1 X D2 backcross, the b/d heterozygote (having the high-affinity Ah receptor) was found to be several times more resistant than the d/d homozygote to a single dose of intraperitoneal ethanol. The D2.B6-Ahb congenic line is also several times more resistant to intraperitoneal ethanol than the B6.D2-Ahb congenic line is also several times more resistant to intraperitoneal ethanol than B6.D2-Ahd congenic line. We found that the waking blood ethanol levels are the same in b/d and d/d mice, suggesting that the relative ethanol resistance in b/d mice cannot be explained on the basis of a difference in central nervous system sensitivity. There are no differences between SS and LS mice or between b/d and d/d mice with regard to (i) blood acetaldehyde levels after a single intraperitoneal dose of ethanol, or (ii) hepatic alcohol dehydrogenase activities. There is a difference in the rate of ethanol elimination: SS more rapid than LS; b/d more rapid than d/d. Although SS mice have lower hepatic aldehyde dehydrogenase activities (cytosolic, mitochondrial low-Km: and mitochondrial high-Km forms) than LS mice, b/d and d/d do not show this difference. These data suggest that a selected mouse breeding program, based on resistance to a single intraperitoneal dose of ethanol, selects concurrently for the hepatic high-affinity Ah receptor. This selective advantage cannot be explained on the basis of changes in alcohol dehydrogenase or aldehyde dehydrogenase activities and might provide insight into the nature of the endogenous ligand for the Ah receptor.

Trout P450IA1: cDNA and deduced protein sequence, expression in liver, and evolutionary significance.

The cytochrome P1(450) (P450IA1) cDNA has been isolated and sequenced from liver of 3-methylcholanthrene-treated rainbow trout (Salmo gairdneri). The cDNA hybridizes to a 2.8-kb mRNA that is induced at least 10-fold by 3-methylcholanthrene. Southern blot analysis suggests the presence of a single gene or a very small number of genes. An open reading frame of the 2573-bp cDNA encodes a 522-residue protein (Mr = 59,241) that is more similar to the mammalian P450IA1 than the mammalian P450IA2 proteins. The aromatic hydrocarbon (Ah) receptor, responsible for mammalian P450IA1 and IA2 inducibility, was detected in trout liver cytosol by specific binding to [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro. Comparison of the fish P450IA1 protein with human, mouse, rat and rabbit P450IA1 and P450IA2 proteins reveals the presence of a remarkably large number of single amino acids and stretches of 2-6 residues in a row that are invariant among these nine P450 proteins. These conserved regions may participate in the binding of the NADPH-P450 oxidoreductase flavoprotein, substrate, or heme.

The murine aromatic hydrocarbon responsiveness locus: a comparison of receptor levels and several inducible enzyme activities among recombinant inbred lines.

The aromatic hydrocarbon responsiveness (Ah) locus has been correlated with genetic differences in the risk of drug toxicity, teratogenesis, chemical carcinogenesis, and mutagenesis. Hepatic cytosolic Ah receptor levels, 2-amino-5-chlorobenzoxazole (zoxazolamine) paralysis time following beta-naphthoflavone treatment and aryl hydrocarbon hydroxylase (AHH3, acetanilide 4-hydroxylase (Ac4H), and NAD(P)H:menadione oxidoreductase (NMOR)4, induction by 3-methylcholanthrene were studied in (a) the progenitors C57BL/6J (Ahb/Ahb) and DBA/2J (Ahd/Ahd) and 25 BXD recombinant inbred lines, (b) the progenitors C57BL/6N and C3H/HeN and 14 B6NXC3N recombinant inbred lines, and (c) the progenitors C57BL/6J and C3H/HeJ and 12 BXH recombinant inbred lines. The Ahb phenotype exhibits greater than 5 femtomole receptor/mg of cytosolic protein, less than or equal to 15 minutes zoxazolamine paralysis time, and twofold to 15-fold induction of these three hepatic enzyme activities; the Ahd phenotype exhibits less than or equal to 2 fmol receptor/mg protein, greater than 15 minutes zoxazolamine paralysis time, and less than 30% induction of these three activities. Among the BXD lines but especially among the B6NXC3N and BXH lines, high frequencies of recombination were found; the phenotype of each of the five parameters did not segregate with the phenotype of each of the other parameters in four or more recombinant lines. This report shows for the first time that AHH induction by 3-methylcholanthrene can occur in the Ahd phenotype mouse. These data underline the complexity of this genetic system when genes from C57BL/6 and DBA/2 are combined and particularly when genes from C57BL/6 and C3H/He inbred mouse strains are combined.

Measurements of the cytosolic Ah receptor among four strains of Drosophila melanogaster.

Four strains of Drosophila melanogaster exhibit differences in aryl hydrocarbon hydroxylase (AHH) inducibility by phenobarbital or Aroclor 1254, yet do not show the typical AHH induction response when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[a]anthracene. Adult flies were nevertheless examined for the presence of cytosolic TCDD-specific binding (Ah receptor). Berlin-K and Haag 79 exhibit AHH induction by Aroclor 1254 and possess detectable amounts of Ah receptor. Hikone-R has negligible AHH inducibility by Aroclor 1254, yet possesses measurable amounts of the receptor. Oregon-K displays AHH induction by Aroclor 1254 but has no detectable levels of the cytosolic receptor. Specific (high-affinity, low-capacity and saturable) binding of [3H-1,6]TCDD to the Ah receptor in D. melanogaster was shown to be similar to that observed in C57BL/6 mouse liver. Similar specific binding of generally labeled [3H]benzo[a]anthracene in D. melanogaster cytosol was not found. These data suggest that the presence of the Ah receptor per se, or quantity of receptor, does not guarantee AHH inducibility by TCDD or benzo[a]anthracene in adults of these four fruit fly strains.

Aryl hydrocarbon hydroxylase induction by benzo[a]anthracene: regulatory gene localized to the distal portion of mouse chromosome 17.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase inducibility by benzo[a]anthracene was studied in 29 somatic cell hybrid clones, developed by fusing mouse spleen or peritoneal cells from four different inbred strains with hypoxanthine phosphoribosyltransferase-deficient Chinese hamster E36 cells. Karyotype analysis plus 25 markers assigned to 16 autosomes and the X chromosome were examined. In 28 of the 29 clones, the presence or absence of inducibility is associated with the presence or absence, respectively, of mouse chromosome 17. Liver microsomal aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene or benzo[a]anthracene was assessed in appropriate backcrosses with the Mus musculus molossinus, M. m. castaneus, MOR/Cv, PL/J, SM/J and DBA/2J inbred strains and in 13 NX8 recombinant inbred lines. Twenty-seven biochemical genetic markers representing all but four autosomes were tested for possible linkage with the hydroxylase inducibility, and no linkage was found. The hepatic Ah receptor was quantitated in 26 BXD recombinant inbred lines; the Ah phenotype did not match exactly any of the more than 70 genes with established strain distribution patterns representing 12 autosomes and at least five unlinked markers. It is concluded that a major gene controlling aryl hydrocarbon hydroxylase inducibility by benzo[a]anthracene is located on chromosome 17. Because there is no significant linkage with any of three biochemical markers in the upper third of the chromosome, we conclude that the inducibility gene is located in the distal 40% of mouse chromosome 17. Whether this trait represents the Ah locus, i.e., the gene encoding the cytosolic Ah receptor, will require further study.

An investigation of genetic variation within a series of congenic strains of mice.

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.

Effects of cimetidine on theophylline, acetaminophen, and zoxazolamine toxicity in the intact mouse.

3-Methylcholanthrene treatment of C57BL/6N mice induces significant amounts of cytochromes P1-450, whereas P1-450 levels in 3-methylcholanthrene-treated DBA/2N mice are no different from those in control C57BL/6N or DBA/2N mice. Comparison of 3-methylcholanthrene-treated C57BL/6N and DBA/2N mice thus provides a convenient means of determining the role of P1-450 metabolism in two strains of mice following identical drug treatment regimens. 3-Methylcholanthrene-induced P1-450 is shown to be more effective than other forms of P-450 in detoxifying theophylline and zoxazolamine and in enhancing the toxicity of acetaminophen. Cimetidine in vivo blocks these metabolic pathways, resulting in increased toxicity of theophylline and zoxazolamine and protection against acetaminophen toxicity. These data illustrate the double-edged sword nature of P1-450 metabolism and the possibility of a paradoxical effect of cimetidine during drug-drug interactions in vivo. Cimetidine is shown to inhibit in vivo and in vitro the metabolism by both 3-methylcholanthrene-induced P1-450 and control forms of P-450; these data suggest that cimetidine may be acting at the level of P-450 reduction by NADPH-P-450 oxidoreductase. This same mechanism of action has been previously suggested for ellipticine.

Biological effects of the Sudan dyes. Role of the Ah cytosolic receptor.

The hepatic induction of two cytochrome P1-450-mediated activities [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (ETR)] was studied following the administration of the azo dyes Sudan I, II, III, and IV. When using Ah-responsive C57BL/6J mice, Sudan dye II proved to be quite potent as an inducer causing almost maximal induction at doses as low as 40 mg/kg (1.4 mumoles/kg body weight); Sudan dyes I, III and IV caused one-half the maximal induction at four times that dose. In contrast, none of these compounds caused induction of AHH or ETR in the Ah-nonresponsive DBA/2J animals. When the dyes were given to B6D2F1 X D2 backcross progeny, a strict correlation with the presence of the Ahb allele and the inducibility of AHH and ETR was observed. When these compounds were examined as agonists for the Ah cytosolic receptor by their capacity to replace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding, Sudan dye II was substantially more effective than Sudan dyes I, III and IV. When four repeated doses of Sudan dye II were administered intraperitoneally to Ah-responsive C57BL/6J mice and Ah-nonresponsive DBA/2J mice, Sudan dye II-induced immunotoxicity was markedly greater in C57BL/6J compared to DBA/2J mice.

Screening of 16 common therapeutic drugs. Possible association with the Ah locus.

16 common therapeutic agents were screened for differences in sedation or lethality between C57BL/6N and DBA/2N inbred mouse strains that had been previously treated with beta-naphthoflavone. No differences were observed for meprobamate, valium, promethazine, valproic acid, lincomycin, imipramine, terbutaline, propoxyphene, nitrofurantoin, amphotericin B, or diphenhydramine. C57BL/6N mice appeared to be more resistant than DBA/2N mice to the lethal effects of isoxsuprine, niridazole, pentazocine, isoniazid, and hydralazine. None of these latter five drugs had any capacity to displace [3H-1,6]2,3,7,8-tetrachlorodibenzo-p-dioxin from the liver cytosolic Ah receptor in C57BL/6N mice. With the use of beta-naphthoflavone-pretreated offspring from the (C57BL/6N) (DBA/2N)F1 X DBA/2N backcross, a strict correlation (100% of 24 individuals in each case) was found between the Ahb allele and resistance to the lethal effects of isoxsuprine or niridazole. No correlation between the Ah locus and pentazocine, hydralazine, or isoniazid lethality was apparent. These results indicate that presence of the Ahb allele is associated with increased protection against isoxsuprine and niridazole lethality. This increased protection may reflect enhanced detoxication metabolic pathways (e.g., induced cytochrome P1-450 and/or uridine diphosphate glucuronosyltransferase controlled by the Ah locus). The increased protection is not related to interaction of these drugs with the Ah receptor. It should be kept in mind that gene-environment interactions involving the Ah locus and isoxsuprine or niridazole may be important in certain clinical instances.

The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor.

The capacity of 19 polycyclic aromatic compounds and 15 benzo[a]pyrene metabolites to displace [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxine ([3H]TCDD) from the mouse liver cytosolic Ah receptor was examined. We compared our data with various parameters taken from previously published results: the capacity of seven polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase (AHH) activity in human cell cultures, the capacity of 10 polycyclic hydrocarbons to induce azo dye N-demethylase activity in rat liver, the capacity of 6 polycyclic hydrocarbons to shorten zoxazolamine paralysis times in the intact rat, and the capacity of 15 benzo[a]pyrene metabolites to induce AHH activity in rat hepatoma H-4-II-E cultures. An excellent correlation is seen between the capacity to displace the radioligand from the Ah receptor and the capacity to induce these monooxygenase activities. differences in the rate of cellular uptake and formation of alkali-extractable metabolites of dibenzo[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene in Hepa-1 mouse hepatoma cell cultures do not account for differences in the capacity of these three polycyclic hydrocarbons to displace [3H]TCDD from the Ah receptor.

Ontogenetic expression of regulatory and structural gene products associated with the Ah locus. Comparison of rat, mouse, rabbit and Sigmoden hispedis.

The murine Ah complex represents a 'cluster' of genes controlling the induction of numerous cytochrome P-450-mediated monooxygenase 'activities' by polycyclic aromatic compounds. These forms of cytochrome represent structural gene products. A major regulatory gene product is a cytosolic receptor to which radiolabeled 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) binds with very high affinity. Developmental differences in mouse, rat and rabbit liver have been previously reported, indicating that some form of temporal control exists during the induction of enzymes associated with the ah locus. The cytosolic receptor levels were determined by sucrose density gradient centrifugation after dextran-charcoal treatment. Hepatic receptor levels, beta-naphthoflavone-inducible and control aryl hydrocarbon hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylase activities and total P-450 content were studied in the Sprague-Dawley rat, C57BL/6N mouse, New Zealand White rabbit and Sigmoden hispedis (cotton rat) as a function of age. 3-Methylcholanthrene-inducible and control aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activities in nonhepatic tissues of the neonatal and adult rabbit were examined. beta-Naphthoflavone-inducible hepatic microsomal proteins detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were also studied in the four species. Taken altogether, the developmental data indicate that: the form of P-450 responsible for induced aryl hydrocarbon hydroxylase activity is not the same as that for induced acetanilide 4-hydroxylase, and neither one is the same as induced cytochrome P-448; the presence of the TCDD-specific cytosolic receptor per se (detected as a distinct high-specificity saturable peak on sucrose density gradients) does not guarantee the expression of inducible aryl hydrocarbon hydroxylase or acetanilide 4-hydroxylase activity; although interesting developmental differences exist among all four species examined, the TCDD-specific receptor is maximal between the neonatal and weaning period, is considerably decreased in the adult, and is suppressed even more during the latter half of pregnancy; in general, the times at which the cytosolic receptor is highest or lowest parallels quite closely the well-known increases in inducible drug-metabolizing enzymes that have been commonly observed in the rat, mouse and other laboratory animals, and more studies are necessary before we understand what the 'TCDD-specific binding peak' (as observed on the sucrose density gradients) actually represents.(ABSTRACT TRUNCATED AT 400 WORDS)

Pyrolysis products from amino acids and protein: highest mutagenicity requires cytochrome P1-450.

Pyrolysis products of proteins and amino acids are highly mutagenic, but metabolism of these chemicals by rat liver subcellular fractions is known to be required for production of the mutagenic intermediates. We examined the mutagenesis of seven purified pyrolysis products from tryptophan, lysine, glutamic acid, and soybean globulin with Salmonella typhimurium strain TA98 in the presence of liver fractions from genetically "responsive" C57BL/6N and Ah(b)/Ah(d) or "nonresponsive" DBA/2N and Ah(d)/Ah(d) mice that had been pretreated in vivo with benzo[a]pyrene. For all pyrolysis products tested, mutagenesis is 2-fold to more than 1000-fold greater with C57BL/6N and Ah(b)/Ah(d) than with DBA/2N or Ah(d)/Ah(d) liver fractions. A sucrose density gradient assay for detecting the Ah regulatory gene product, the receptor, was studied with C57BL/6N hepatic cytosol. At levels 100 times in excess of [1,6-(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin, nonlabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphthoflavone (inducers of cytochrome P(1)-450) are able to displace the radioligand from its hepatic cytosolic receptor; four pyrolysates from tryptophan, glutamic acid, and soybean globulin did not have this capacity. These data indicate that the pyrolysis products tested, although not effective as inducers of cytochrome P(1)-450, are most mutagenic when metabolized by P(1)-450. Potent P(1)-450 inducers-present in pyrolysates during the combustion process-might be present in quantities insufficient to initiate mutagenesis or carcinogenesis but might have a synergistic action, or act as "comutagens" or "cocarcinogens," with the N-containing heterocyclic pyrolysis products. A quantitative relationship between mutagenic and carcinogenic potency of these pyrolysis products remains, however, to be demonstrated.