Developmental effects of petroleum creosote on mice following oral exposure.

Petroleum creosote, dissolved in dimethyl sulfoxide, was administered by gavage to pregnant ICR mice on days 5-9 of gestation at a single dose (400 mg/kg body weight per day). Animals were euthanized on day 17 of gestation, and live fetuses were weighed and examined for skeletal and visceral malformations. Maternal body weights were significantly lowered in both the group administered creosote and the group administered the solvent alone. The number of live fetuses, dead fetuses, resorptions, and the sex ratio in the live fetuses were similar in all groups. Petroleum creosote as administered in this study was not found to be teratogenic in ICR mice.

In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture.

A mouse preimplantation embryo culture system was utilized to characterize the in vitro embryotoxicity of petroleum creosote (PC), a complex mixture of aliphatic and polycyclic aromatic hydrocarbons. ICR mouse embryos, collected on d 3.5 of gestation (blastocyst stage), were exposed for 1 h to varying concentrations of petroleum creosote in serum-supplemented culture medium. Parallel embryo cultures were exposed to PC in medium supplemented with rodent hepatic S9 microsomal fractions to monitor the role of bioactivation in PC-induced embryotoxicity. Embryos were subsequently cultured in control medium for 72 h and observed for viability as well as specific, time-dependent developmental end points--hatching and attachment to the culture dish at 48 h, and trophoblastic outgrowth with a distinct inner cell mass at 72 h. Embryonic viability varied in inverse proportion to PC concentration. Petroleum creosote caused embryolethal effects at concentrations of 33 micrograms/ml of culture medium and 54 micrograms/ml. Embryotoxicity was not observed at 22 micrograms/ml. Culture supplementation with rodent hepatic S9 fractions did not modify, either qualitatively or quantitatively, the embryotoxicity of PC in vitro. These findings implicate PC as a prenatal toxicant and support environmental and human health concerns regarding PC exposure from PC-containing chemical waste sites.

Role of biotransformation in the in vitro preimplantation embryotoxicity of naphthalene.

The in vitro developmental toxicity of the bicyclic aromatic hydrocarbon naphthalene was characterized with a preimplantation mouse embryo culture system. Day 3 ICR mouse blastocysts were co-cultured with naphthalene for 1 h either alone or in media supplemented with an Aroclor-induced rat S-9 preparation and cofactors. Toxin-treated blastocysts were subsequently cultured in NCTC 109 media with 10% fetal bovine serum for 72 h to observe the developmental effects of exposure. Developmental parameters observed included viability, hatching, culture dish attachment and trophoblastic outgrowth with the presence of a distinct inner cell mass. At media concentrations up to 0.78 mM, naphthalene alone exhibited negligible toxic effects in culture; however naphthalene co-cultured with Aroclor-induced rat hepatic S-9 fractions exhibited concentration-dependent embryolethality with an approximate LC50 of 0.18 mM in media. Naphthalene also induced concentration-dependent embryotoxicity at all observed parameters in S-9-supplemented media at concentrations ranging from 0.20 to 0.78 mM. These findings document the role of biotransformation in naphthalene's embryotoxicity to early mouse blastocysts and implicate naphthalene as a potentially embryotoxic and abortifacient component of polycyclic aromatic hydrocarbon mixtures.

Microsomal activation of constituents of white snakeroot (Eupatorium rugosum Houtt) to form toxic products.

Components of white snakeroot, a plant toxic to livestock and human beings, were activated by Aroclor 1254-induced rat liver microsomes. The toxic products of microsomal activation were evaluated in murine melanoma (B16F1) cell cultures. Toxic products in white snakeroot were inactive in cell culture systems without microsomal activation. This activation system revealed that at least 2 fractions of white snakeroot were metabolically activated to cytotoxic agents. The autocatalytic inactivator of cytochrome P-450, 1-aminobenzotriazole, inhibited activation of white snakeroot constituents by rat liver microsomes.

Dietary aflatoxins, intelligence and school performance in southern Georgia.

In a region of southern Georgia known for poor school performance, the mothers of the mentally retarded children had diets that differed from the average in terms of foods, but did not differ in terms of critical nutrients. The consumption of large amounts of corn, rice, peanuts and milk (foods potentially high in aflatoxins) was significantly related to mental retardation of children in one county having high levels of aflatoxins in the food supply. However, no such relationship was found in a county having trivial levels of aflatoxins in the diet.

Formation and persistence of sterigmatocystin--DNA adducts in rat liver determined via 32P-postlabeling analysis.

A 32P-postlabeling method has been employed to detect the in vitro and in vivo modification of DNA by the mycotoxin sterigmatocystin (ST). ST-modified DNA was initially incubated under buffered alkaline conditions to convert unstable ST-N7-guanine moieties to stable, putative ST-formamidopyrimidine derivatives. DNA was subsequently digested with micrococcal nuclease and spleen phosphodiesterase, and the resulting ST-modified nucleotides, purified by reverse-phase thin-layer chromatography (TLC), were labeled at the 5' position via incubation with [gamma-32P]ATP and T4 polynucleotide kinase. 32P-labeled ST-nucleotides were separated by reverse-phase and anion-exchange TLC. Cerenkov quantitation of excised TLC fractions indicated that ST-DNA moieties could be detected with a sensitivity of 1 ST adduct in 3-5 X 10(7) nucleotides. Initial enzymatic digestion of ST-modified DNA was found to yield ST-modified di- and trinucleotides which, upon 32P-labeling followed by incubation with nuclease P1, liberated unmodified 5'-terminal nucleotides suggesting that ST-formamidopyrimidine-modified DNA was a poor substrate for micrococcal nuclease and spleen phosphodiesterase. Dose-dependent ST-DNA adduct formation was detected in the liver of male Fischer 344 rats over a 27-fold range of ST administered (0.33-9 mg/kg). In addition, ST-DNA adducts, formed in rats given a 9 mg/kg dose, were found to persist up to 105 days after treatment at a level of 0.5% of the 2-h value. Loss of these adducts from liver DNA was observed to exhibit a triphasic profile: rapid loss during the first 24 h (t 1/2 = 12 h) followed by a slower decline from 1 to 14 days post dosing (t 1/2 = 7 days) and an extremely slow decline from days 14 to 105 post treatment (t 1/2 = 109 days). This experimental approach to the study of mycotoxin-DNA interactions permits the quantitative description of DNA modification in ST-treated animals. Further refinement of this approach may be useful in defining the precise relationship between ST exposure and tumorigenesis in ST-exposed human populations.

Quantitative and qualitative characterization of aflatoxin B1 adducts formed in vivo within the ribosomal RNA genes of rat liver DNA.

We have examined the time course and patterns of covalent aflatoxin B1:DNA adducts produced within the ribosomal RNA gene sequences isolated from the liver nuclear DNA of aflatoxin B1-treated animals. Liver nuclear DNA was initially enriched in ribosomal DNA by cesium salt density centrifugation, and incubated under alkaline conditions to stabilize bound aflatoxin B1-DNA moieties. Alkali-treated DNA was hybridized to 18S and 28S rRNA, and the RNA:DNA hybrids were recovered by cesium chloride centrifugation. Ribosomal DNA sequences within nuclear DNA were found to be preferential targets for aflatoxin B1 modification. Over a 12-h period after administration of 1-mg [3H]aflatoxin B1/kg dose ribosomal DNA contained 4 to 5 times more aflatoxin B1 residues per mg DNA than did total nuclear DNA. Aflatoxin B1 residues bound to ribosomal DNA were also found to be removed more rapidly than from total nuclear DNA by a factor of 5.7 over the 12-h period postdosing. Levels of the principal aflatoxin B1 adduct, 2,3-dihydro-3-hydroxy(N7-guanyl)aflatoxin B1, as well as the stable formamidopyrimidine derivatives of the parent adduct were also determined. Nuclear DNA isolates were heated to induce depurination of the principal N7-guanine adduct, and differences in adduct levels between alkali-treated (stabilized) and depurinated DNA samples were taken as an approximation of initial levels of this aflatoxin B1:DNA moiety in ribosomal isolates. No differences in the proportions of these aflatoxin B1:DNA adduct species were found in ribosomal as compared to total nuclear DNA, and we conclude that the preferential formation and removal of aflatoxin B1:DNA moieties within ribosomal DNA is not associated with a pattern of adducts qualitatively different from that in total nuclear DNA.

DNA polymerase activity in a repair-deficient human cell line.

A human low-density-lipoprotein (LDL) receptor-deficient diploid fibroblast cell line (GM1915) was determined to be short patch competent (DNA polymerase-beta) and long patch deficient (DNA polymerase-alpha) for DNA excision repair. Analysis of DNA from GM1915 cells or from WI38 control cells, following treatment with a mutagen known to initiate long patch excision repair, showed that GM1915 cells exhibited decreased resynthesis of oligonucleotide segments excised during repair. When cells deficient in DNA polymerase-alpha activity were permeabilized to permit LDL entry, repair synthesis immediately increased. These data suggest that DNA polymerase-alpha is not activated by mutagen treatment in GM1915 cells and that introduction of LDL into the cells results in activation of the enzyme.

Aflatoxin B1 metabolism in the rat: polyhalogenated biphenyl enhanced conversion to aflatoxin M1.

The effects of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) on the formation in vitro of aflatoxin Q1 and aflatoxin M1 from aflatoxin B1 by rat-liver microsomes were investigated. AFB1 metabolism by hepatic microsomes from PBB- and PCB-treated rats resulted in 16- and 30-fold increases, respectively, in levels of aflatoxin M1. The enhanced formation of aflatoxin M1 did not correlate with PBB and PCB stimulation of benzo[a]pyrene hydroxylase (AHH) activity. Studies in vivo clearly demonstrated enhanced secretion of aflatoxin M1 by female lactating rats with prior exposure to PCBs. PCB pretreatment enhanced the activity of mammary as well as hepatic tissue microsomal preparations in converting aflatoxin B1 to aflatoxin M1. Our findings indicate that PCB exposure increases the production of aflatoxin M1 in vitro and also increases the levels of aflatoxin M1 released into the milk.

Quantitation of aflatoxin B1 adduction within the ribosomal RNA gene sequences of rat liver DNA.

The in vivo formation of covalent aflatoxin B1 (AFB1)-DNA adducts within the rRNA gene sequences of nuclear DNA has been studied in AFB1-treated rats. Liver nuclear DNA, enriched in ribosomal DNA (rDNA) by one round of cesium salt density gradient centrifugation, was treated under buffered alkaline conditions to convert unstable AFB1-N7-guanine adducts to stable AFB1-formamidopyrimidine derivatives. The alkali-treated DNA was hybridized to 18S and 28S rRNA in 70% formamide buffer to form rRNA X rDNA hybrids. These hybrids were separated from the bulk of nuclear DNA by two rounds of centrifugation in CsCl, and the level of AFB1 adduction to rDNA versus total nuclear DNA was compared as a function of dose 2 hr after AFB1 administration. Over an 8-fold dose range (0.25-2.0 mg of AFB1 per kg of body weight), rDNA contained 4- to 5-fold more AFB1 residues than nuclear DNA, indicating that rDNA is preferentially accessible to carcinogen modification in vivo. While aflatoxin B1 forms adducts with DNA principally at guanine residues, the guanine enrichment of rDNA was insufficient to explain the magnitude of observed preferential AFB1 modification of rDNA. These results support the hypothesis that rDNA regions are preferentially accessible to carcinogen modification because of the diffuse conformation maintained within transcribed genes. This experimental approach permits the quantitative description of carcinogen modification within a defined gene sequence; further refinement of this approach may be useful in defining the precise relationships between covalent chemical-DNA interactions and the alterations in gene expression that result.