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Six TiO₂ and two CeO₂ nanomaterials with dry sizes ranging from 6-410 nm were tested for their ability to cause DNA centered free radicals in vitro in the concentration range of 10-3,000 ug/ml. All eight of the nanomaterials significantly increased the adduction of the spin trap agent 5,5-dimethyl-1-pyroline N-oxide (DMPO) to DNA as measured by the experimental technique of immuno-spin trapping. The eight nanomaterials differed considerably in their potency, slope, and active concentration. The largest increase in DNA nitrone adducts was caused by a TiO₂ nanomaterial (25 nm, anatase) from Alfa Aesar. Some nanomaterials that increased the amount of DNA nitrone adducts at the lowest exposure concentrations (100 ug/ml) were Degussa TiO₂ (31 nm), Alfa Aesar TiO₂ (25 nm, anatase) and Nanoamor CeO₂ (8 nm, cerianite). At exposure concentrations of 10 or 30 ug/ml, no nanomaterials showed significant in vitro formation of DNA nitrone adducts.

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis. Fil: Gomez-Mejiba, Sandra Esther. Oklahoma Medical Research Foundation; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zhai, Zili. Oklahoma Medical Research Foundation; Estados Unidos Fil: Gimenez, Maria Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis; Argentina Fil: Ashby, Michael T.. University of Oklahoma; Estados Unidos Fil: Chilakapati, Jaya. United States Environmental Protection Agency. National Health and Environmental Effects Research; Estados Unidos Fil: Kitchin, Kirk. United States Environmental Protection Agency. National Health and Environmental Effects Research; Estados Unidos Fil: Mason, Ronald P.. National Institutes of Health; Estados Unidos Fil: Ramirez, Dario. Oklahoma Medical Research Foundation; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

111 chemicals of known rodent carcinogenicity (49 carcinogens, 62 noncarcinogens), including many promoters of carcinogenesis, nongenotoxic carcinogens, hepatocarcinogens, and halogenated hydrocarbons, were selected for study. The chemicals were administered by gavage in two dose levels to female Sprague-Dawley rats. The effects of these 111 chemicals on 4 biochemical assays (hepatic DNA damage by alkaline elution (DD), hepatic ornithine decarboxylase activity (ODC), serum alanine aminotransferase activity (ALT), and hepatic cytochrome P-450 content (P450) were determined. Composite parameters are defined as follows: CP = [ODC and P450), CT = [ALT and ODC), and TS = [DD or CP or CT]. The operational characteristics of TS for predicting rodent cancer were sensitivity 55%, specificity 87%, positive predictivity 77%, negative predictivity 71%, and concordance 73%. For these chemicals, the 73% concordance of this study was superior to the concordance obtained from published data from other laboratories on the Ames test (53%), structural alerts (SA) (46%), chromosome aberrations in Chinese hamster ovary cells (ABS) (48%), cell mutation in mouse lymphoma 15178Y cells (MOLY) (52%), and sister-chromatid exchange in Chinese hamster ovary cells (SCE) (60%). The 4 in vivo biochemical assays were complementary to each other. The composite parameter TS also shows complementarity to all 5 other predictors of rodent cancer examined in this paper. For example. the Ames test alone has a concordance of only 53%. In combination with TS, the concordance is increased to 62% (Ames or TS) or to 63% (Ames and TS). For the 67 chemicals with data available for SA, the concordance for predicting rodent carcinogenicity was 47% (for SA alone), 54% (for SA or TS), and 66% (for SA and TS). These biochemical assays will be useful: (1) to predict rodent carcinogenicity per se, (2) to ‘confirm’ the results of short-term mutagenicity tests by the high specificity mode of the biochemical assays (the specificity and positive predictivity are both 100%), and (3) to be a component of future complementary batteries of tests for predicting rodent carcinogenicity.

Exposure to inorganic arsenic (iAs) induces cancer in human lungs, urinary bladder, skin, kidney, and liver, with the majority of deaths from lung and bladder cancer. To date, cancer risk assessments for iAs have not relied on mechanistic data, as we have lacked sufficient understanding of arsenic's pharmacokinetics and mode(s) of carcinogenic action (MOA). Furthermore, while there are vast amounts of toxicological data on iAs, relatively little of it has been collected using experimental designs that efficiently support development of biologically based dose-response (BBDR) models and subsequently risk assessment. This review outlines an efficient approach to the development of a BBDR model for iAs that would reduce uncertainties in its cancer risk assessment. This BBDR-based approach is illustrated by using oxidative stress as the carcinogenic MOA for iAs but would be generically applicable to other MOAs. Six major research needs that will facilitate BBDR model development for arsenic-induced cancer are (1) MOA research, which is needed to reduce the uncertainty in risk assessment; (2) development and integration of the pharmacodynamic component (MOA) of the BBDR model; (3) dose-response and extrapolation model selection; (4) the determination of internal human speciated arsenical concentrations to improve physiologically based pharmacokinetic (PBPK) models; (5) animal models of arsenic carcinogenesis; and (6) the determination of the low dose human relationship for death from cancer, particularly in lungs and urinary bladder. The major parts of the BBDR model are arsenic exposure, a physiologically based pharmacokinetic model, reactive species, antioxidant defenses, oxidative stress, cytotoxicity, growth factors, transcription factors, DNA damage, chromosome damage, cell proliferation, mutation accumulation, and cancer. The BBDR model will need to be developed concurrently with data collection so that model uncertainties can be identified and addressed through an iterative process of targeted additional research.

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips®. Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:406–418, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20304

Lung is a major target for arsenic carcinogenesis in humans by both oral and inhalation routes. However, the carcinogenic mode of action of arsenicals is unknown. We investigated the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII) and dimethylthioarsinic acid (DMTA), a sulfur containing dimethyl arsenic metabolite, in human bronchial epithelial (BEAS-2B) cells. Cells were exposed to 3, 15 microM-iAsIII; 0.3, 1 microM-MMAIII; 0.2, 1 microM-DMAIII; 0.2, 0.9 microM-DMTA as non-cytotoxic and minimally cytotoxic ( approximately 20%) concentrations based on Neutral Red uptake assays after 24h of culture. Total RNA was isolated and gene expression analysis conducted using Affymetrix Human Genome 133 Plus 2.0 arrays. Differentially expressed genes (DEGs) were determined using a one-way ANOVA (por =0.05) by Rosetta Resolver, a Benjamini-Hochberg FDR (false discovery rate) multiple testing correction (0.05) followed by a Scheffe's post hoc test. For all compounds except DMTA,90% of DEG altered in the low concentration were also changed at the high concentration. There was a clear dose-response seen in the number of DEGs for all four compounds. iAsIII showed the highest number of DEG at both concentrations (2708 and 123, high and low, respectively). 1749, 420 and 120 DEGs were unique to the high concentrations of iAsIII, MMAIII and DMAIII, respectively. Transferrin receptor is a common DEG in low concentration arsenical treated cells. Ingenuity Pathway Analysis revealed p53 signaling (E2F1 and 2, SERPIN), and cell cycle related genes (cyclin D1) were altered by the high concentrations of DMTA, MMAIII and iAsIII. Oxidative stress (DUSP1, GPX2, NQO1, GCLC) and NF-kappaB signaling (TLR4, NF-kappaB) pathways were changed by the high concentrations of MMAIII and iAsIII. The genes identified in this study can be a valuable tool to determine the mechanism of arsenic toxicity and cancer formation. A number of similarities were observed in the gene expression profiles of DMAIII and DMTA and also iAsIII and MMAIII. These findings reveal some biological effects of arsenicals that will aid in creating a better risk assessment model for arsenical-induced lung cancer.

Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (approximately 8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.

The effects of monomethylarsonous acid (MMA[III]) and arsenite, administered in drinking water on tissue levels of arsenicals, cytogenetics, and mouse skin tumorigenicity were determined. A low-methionine diet modified the pattern of arsenical tissue concentrations and decreased the tissue arsenical concentrations, particularly in kidney and urinary bladder, less so in liver, and had little effect in the lungs. In mice given 75 ppm arsenite and a low-methionine diet, the urinary bladder tissue levels were only 29%, 26%, and 38% of the inorganic arsenic (iAs), MMA, and dimethylarsinic acid (DMA) concentrations found in mice eating the control diet. In K6/ODC transgenic mice that consumed a normal diet (Purina 5002), a 26-week drinking water exposure to 10 ppm arsenite resulted in 5% of the treated animals having squamous skin tumors. Exposure to 10, 50, 75, or 150 ppm MMA(III) caused 5%, 6.7%, 5%, or 0% tumor-bearing animals. A low-methionine diet did not markedly change the incidence of skin tumors--10 ppm arsenite led to 10% tumors. With a low-methionine diet, 10 and 50 ppm, MMA(III) caused 5% and 6.7% tumor-bearing animals. In comparing the frequency of tumors in the concurrent control groups (1/70, 1.4%) with the frequency of tumors in the pooled arsenical-treated responsive groups (8/122, 6.6%), there is an excess of 6 mouse skin tumors observed in the pooled arsenical-responsive treatment groups compared to the expected number of tumors based on frequency of tumors observed in concurrent control mice. In summary, studies with MMA(III) and arsenite-treated K6/ODC transgenic mice showed (1) a low-methionine diet substantially altered mouse tissue arsenical levels and (2) numerically elevated incidence of mouse skin tumors following arsenical exposures.

A large amount of evidence suggests that arsenicals act via oxidative stress in causing cancer in humans and experimental animals. It is possible that arsenicals could bind in situ close to nuclear DNA followed by Haber-Weiss type oxidative DNA damage. Therefore, we tested this hypothesis by using radioactive (73)As labeled arsenite and vacuum filtration methodology to determine the binding affinity and capacity of (73)As arsenite to calf thymus DNA and Type 2A unfractionated histones, histone H3, H4 and horse spleen ferritin. Arsenicals are known to release redox active Fe from ferritin. At concentrations up to about 1 mM, neither DNA nor any of the three proteins studied, Type II-A histones, histone H3, H4 or ferritin, bound radioactive arsenite in a specific manner. Therefore, it appears highly unlikely that initial in situ binding of trivalent arsenicals, followed by in situ oxidative DNA damage, can account for arsenic's carcinogenicity. This experimental evidence (lack of arsenite binding to DNA, histone Type II-A and histone H3, H4) does not rule out other possible oxidative stress modes of action for arsenic such as (a) diffusion of longer lived oxidative stress molecules, such as H(2)O(2) into the nucleus and ensuing oxidative damage, (b) redox chemistry by unbound arsenicals in the nucleus, or (c) arsenical-induced perturbations in Fe, Cu or other metals which are already known to oxidize DNA in vitro and in vivo.

Chronic drinking water exposure to inorganic arsenic and its metabolites increases tumor frequency in the skin of K6/ODC transgenic mice. To identify potential biomarkers and modes of action for this skin tumorigenicity, we characterized gene expression profiles from analysis of K6/ODC mice administered 0, 0.05, 0.25, 1.0 and 10 ppm sodium arsenite in their drinking water for 4 weeks. Following exposure, total RNA was isolated from mouse skin and processed to biotin-labeled cRNA for microarray analyses. Skin gene expression was analyzed with Affymetrix Mouse Genome 430A 2.0 GeneChips®, and pathway analysis was conducted with DAVID (NIH), Ingenuity® Systems and MetaCore's GeneGo. Differential expression of several key genes was verified through qPCR. Only the highest dose (10 ppm) resulted in significantly altered KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, including MAPK, regulation of actin cytoskeleton, Wnt, Jak–Stat, Tight junction, Toll-like, phosphatidylinositol and insulin signaling pathways. Approximately 20 genes exhibited a dose response, including several genes known to be associated with carcinogenesis or tumor progression including cyclin D1, CLIC4, Ephrin A1, STAT3 and DNA methyltransferase 3a. Although transcription changes in all identified genes have not previously been linked to arsenic carcinogenesis, their association with carcinogenesis in other systems suggests that these genes may play a role in the early stages of arsenic-induced skin carcinogenesis and can be considered potential biomarkers.

Seafood frequently contains high concentrations of arsenic (approximately 10-100 mg/kg dry weight). In marine algae (seaweed), this arsenic occurs predominantly as ribose derivatives known collectively as arsenosugars. Although it is clear that arsenosugars are not acutely toxic, there is a possibility of arsenosugars having slight chronic toxicity. In general, trivalent arsenicals are more toxic than their pentavalent counterparts, so in this work we examine the hypothesis that trivalent arsenosugars might be significantly more toxic than pentavalent arsenosugars in vitro. We compared the in vitro toxicity of (R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-beta-D-riboside, a pentavalent arsenosugar, to that of its trivalent counterpart, (R)-2,3-dihydroxypropyl-5-deoxy-5-dimethylarsino-beta-D-riboside. The trivalent arsenosugar nicked plasmid DNA, whereas the pentavalent arsenosugar did not. The trivalent arsenosugar was more cytotoxic (IC50 = 200 microM, 48 h exposure) than its pentavalent counterpart (IC50 > 6000 microM, 48 h exposure) in normal human epidermal keratinocytes in vitro as determined via the neutral red uptake assay. However, both the trivalent and the pentavalent arsenosugars were significantly less toxic than MMA(III), DMA(III), and arsenate. Neither the pentavalent arsenosugar nor the trivalent arsenosugar were mutagenic in Salmonella TA104. The trivalent arsenosugar was readily formed by reaction of the pentavalent arsenosugar with thiol compounds, including, cysteine, glutathione, and dithioerythritol. This work suggests that the reduction of pentavalent arsenosugars to trivalent arsenosugars in biology might have environmental consequences, especially because seaweed consumption is a significant environmental source for human exposure to arsenicals.

Publisher Summary Chemical properties (soft acid and sulfhydryls, nucleophilicity, free radicals and ROS, atomic and molecular orbitals, electronegativity, valence state, enthalpies, and ionization energies) can explain only a part of the biological activity of arsenicals. Interaction studies between various arsenicals and the appropriate target biological molecules can be expected to be much more fruitful. It may eventually be found that one or more of the chemical properties of arsenicals may enable arsenic (As) species to act through several modes of carcinogenic action at many stages of multistage carcinogenesis. Important future research questions include (1) which or how many of the known As chemical species are causally active in toxicity/carcinogenesis and (2) is there a single predominant mode or mechanism of As toxicity/carcinogenesis or do many modes/mechanisms participate. Identifying biological targets that respond at low-tissue concentrations of arsenicals is a matter of great importance. A scientific and risk assessment issue of major importance is how one should mathematically extrapolate from the higher arsenic exposure concentrations associated with known toxicity and carcinogenicity to the lower arsenic concentration region below that in which human epidemiology and experimental biological experimentation can give useful data.

The in vitro effects of four different species of arsenic (arsenate, arsenite, monomethylarsonic acid, and dimethylarsinic acid) in mobilizing iron from horse spleen ferritin under aerobic and anaerobic conditions were investigated. Dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) significantly released iron from horse spleen ferritin either with or without the presence of ascorbic acid, a strong synergistic agent. Ascorbic acid-mediated iron release was time-dependent as well as both DMA(III) and ferritin concentration-dependent. Iron release from ferritin by DMA(III)) alone or with ascorbic acid was not significantly inhibited by superoxide dismutase (150 or 300 units/ml). However, the iron release was greater under anaerobic conditions (nitrogen gas), which indicates direct chemical reduction of iron from ferritin by DMA(III), with or without ascorbic acid. Both DMA(V) and DMA(III)) released iron from both horse spleen and human liver ferritin. Further, the release of ferritin iron by DMA(III)) with ascorbic acid catalyzed bleomycin-dependent degradation of calf thymus DNA. These results indicate that exogenous methylated arsenic species and endogenous ascorbic acid can cause (a) the release of iron from ferritin, (b) the iron-dependent formation of reactive oxygen species, and (c) DNA damage. This reactive oxygen species pathway could be a mechanism of action of arsenic carcinogenesis in man.

Rat heme oxygenase (HO) activity was used as a specific (among forms of arsenic) and sensitive biomarker of effect for orally administered sodium arsenite in rats. Time course studies showed that HO was induced in rat liver from 2 to 48 h in both rat liver and kidney. Hepatic and renal inorganic arsenic (iAs) concentrations were high at times preceding a high degree of HO induction. At times following pronounced HO induction, tissue dimethylarsinic acid concentrations were high. Dose-response studies of arsenite showed substantial HO induction in liver at doses of 30 micromol/kg and higher and in the kidney at doses of 100 micromol/kg and higher. Doses of 10 (in liver) and of 30 micromol/kg (in kidney) sodium arsenite given by gavage did not significantly induce rat HO activity. Speciation of tissue total arsenic into iAs, methylarsonic acid (MMA), and dimethylarsinic acid (DMA) permits us to link tissue iAs and HO enzyme induction. There was a linear relationship between tissue inorganic arsenic (iAs) concentration and tissue HO in individual rats (r(2) = 0.780 in liver and r(2) = 0.797 in kidney). Nonlinear relationships were observed between administered arsenite dose and either liver or kidney iAs concentration. Overall, there was a sublinear relationship between administered arsenite and biological effect in rats. Teratogenesis Carcinog. Mutagen. 19:385-402, 1999. Published 1999 Wiley-Liss, Inc.

This study examined the potential use of hemoglobin (Hb)- and serum-protein adducts of alachlor as potential biomarkers of alachlor exposure, a genotoxic and carcinogenic herbicide. The method developed was based on the observation that cleavage of S-cysteinyl alachlor-protein adducts by methanesulfonic acid gave the rearrangement product 3-(2',6'-diethylphenyl)-1, 3-thiazolidine-4-one (TZO). The structure of TZO was confirmed by mass spectroscopy, NMR spectroscopy, and independent synthesis. In the assay, treatment of alachlor-cysteinyl protein adducts by methanesulfonic acid was followed by extraction and analysis. TZO was detected and quantitated by electron-impact GC/MS in the single ion-monitoring mode. [ring-13C6]Alachlor-N-acetylcysteine was added as an internal standard prior to treatment and was converted to [ring-13C6]TZO, allowing response factors to be used to quantitate TZO concentrations. Incubations of alachlor (0-1000 microM) with human albumin and bovine serum albumin (BSA) resulted in linear adduct formation with both proteins. Maximal adduction levels of 613-1130 pmol alachlor-albumin adducts/mg protein were observed, with BSA binding close to twice that of human albumin. A linear concentration response of alachlor-Hb adducts was observed when whole blood from female CD rats was incubated with alachlor in vitro at concentrations up to 300 microM. Maximal binding was 1860 pmol alachlor-Hb adducts/mg globin. Male CD rats treated with alachlor at 150 mg/kg body wt/day ip for 0, 1, 2, and 3 days were sacrificed 4 days after final dosing. A maximal binding of 2250 pmol alachlor-Hb adducts/mg globin was observed. This assay provides a new approach for biomonitoring alachlor levels in experimental animals and has the potential for use in humans.

A combination of known biochemical techniques (binding of bromocresol green to human albumin, nondenaturing electrophoresis and electroelution) have been utilized in a novel purification of human albumin. This paper reports (a) purification of intact, nondenatured human albumin, (b) simultaneous purification of albumin and hemoglobin from a human plasma and red blood cell lysate mixture, (c) development of a purification method for the two most commonly employed protein biomarkers in human environmental epidemiology and (d) demonstration of the general technique of protein purification of colored ligand-protein complexes by nondenaturing electrophoresis. The noncovalent binding between a protein (albumin) and a colored ligand (bromocresol green) is the biochemical characteristic exploited in this novel purification scheme. This general purification method may be useful for other colored ligand or fluorescent ligand binding proteins. For small-scale electrophoresis, the amount of protein isolated, percentage yield and protein purity (estimated by SDS-PAGE) were 270 micrograms, 80% yield and99% purity for albumin and 217 micrograms, 54% yield and99% purity for hemoglobin, respectively. For large-scale electrophoresis the comparable data was 38.3 mg, 57% yield and 98% purity for albumin and 17.2 mg, 57% yield and 99% purity for hemoglobin, respectively.

In a previous study, we found that sodium arsenite increased hepatic ornithine decarboxylase (ODC) activity and hepatic heme oxygenase (HO) activity, but did not cause any DNA damage in adult female rat liver or lung, suggesting that arsenite may be a promoter of carcinogenesis. In this study sodium arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) were administered orally in equitoxic doses to adult female rats at 21 and 4 h prior to sacrifice. DNA damage (DD), cytochrome P450 content (P450), glutathione content (GSH), ODC, serum alanine aminotransferase (ALT) and HO were measured in liver and/or lung tissue. At 60 mg/kg in rat liver, sodium arsenate increased hepatic HO fivefold. MMA decreased ALT at 226 mg/kg, decreased ALT and GSH at 679 mg/kg and also increased P450 at 679 mg/kg in rat liver. DMA decreased ALT and hepatic GSH and increased hepatic HO at 387 mg/kg. In the lung, DMA decreased ODC at both 129 and 387 mg/kg. DD in lung tissue was significantly higher at 387 mg/kg DMA, demonstrating organ specific DNA damage. The biochemical effects and the inferred oncologic potential of the four major forms of arsenic (arsenate, arsenite, MMA and DMA) differ dramatically. The inorganic forms (arsenate and arsenite) are similar to each other (both good HO inducers); the methylated organic forms of arsenic (MMA and DMA) also share a similar pattern of biochemical effects (decreased GSH and ALT, increased P450). All six of the biochemical parameters studied were altered by DMA in either rat liver or lung.

An experimental approach was taken to the question of dose-response curves for chemical carcinogenesis, using DNA damage as a biomarker. Female rats were give 13 different doses of 1,2-dimethylhydrazine (from 1.4 to 135,000 micrograms/kg) and the subsequent hepatic DNA damage was determined by the alkaline elution technique. DMH doses below 450 micrograms/kg did not significantly damage DNA; all DMH doses of 1000 micrograms/kg or higher damaged rat hepatic DNA (P0.05). In this study the x values (dose) ranged over five orders of magnitude and the y values (DNA damage) ranged 30-fold. Ten different regression models (linear, quadratic, cubic, power, and six nonlinear transition models) were compared in their ability to fit the experimental data. With respect to log transformed dose, the six nonlinear transition equations fit the data considerably better than the four power type of equations. A sigmoid model fit to the log transformed dose of 1,2-dimethylhydrazine had an r2 of 0.9979, a degree of freedom adjusted r2 of 0.9969, a F-statistic of 1,457, and a fit standard error of 0.50. With respect to untransformed dose, only three equations (sigmoid, cascade and gaussian cumulative) could creditably fit the DMH data. The experimental results are interpreted with respect to hormesis, use of log transformed dose, sigmoid dose-response models, thresholds of biological response and cancer risk assessment.

Sodium arsenite and cadmium chloride, were administered orally to adult female rats at 21 and 4 h prior to sacrifice. Liver, lung, skin and urinary bladder were the tissues studied. DNA damage, cytochrome P450, glutathione content (GSH), ornithine decarboxylase (ODC), serum alanine aminotransferase and heme oxygenase activity were measured. Sodium arsenite increased rat hepatic ODC activity at 1.6 and 24.6 mg/kg and hepatic heme oxygenase activity at 8.2 and 24.6 mg/kg, but did not cause any DNA damage. Cadmium chloride did not affect any of the six parameters tested. These findings suggest that sodium arsenite may be a promoter rather than an initiator of carcinogenesis.

An experimental approach was taken to the question of dose-response curves for chemical carcinogenesis. DNA damage in female rat liver was chosen as the experimental parameter because all chemicals found to damage hepatic DNA were rodent carcinogens. The lowest dose causing DNA damage was determined for the 12 active chemicals (1,2-dibromoethane, 1,2-dibromo-3-chloropropane, 1,2-dichloroethane, 1,4-dioxane, methylene chloride, auramine O, Michler's ketone, selenium sulfide, 1,3-dichloropropene, 1,2-dimethylhydrazine, N-nitroso-piperidine and butylated hydroxytoluene). The resulting dose-response curves for rat hepatic DNA damage were plotted versus log of the molar dose (all activity was in five orders of magnitude) and versus percent of chemicals' oral rat LD50 (most of the activity was in only two orders of magnitude). Dose-response studies of the active chemicals were analyzed by regression methods. With the exception of butylated hydroxytoluene, the dose-response curves fit a linear model well (r2 = 0.886) and a quadratic model even better (r2 = 0.947). Based on experimental data from 11 DNA-damaging carcinogens (a dose range of 6 orders of magnitude), an equation and graph of the dose-response relationship of an 'average DNA-damaging carcinogen' is presented over the x-axis dose range of eight orders of magnitude.

Twenty-one chemicals carcinogenic in rodent bioassays were selected for study. The chemicals were administered by gavage in two dose levels to female Sprague-Dawley rats. The effects of these 21 chemicals on four biochemical assays [hepatic DNA damage by alkaline elution (DD), hepatic ornithine decarboxylase activity (ODC), serum alanine aminotransferase activity (ALT), and hepatic cytochrome P-450 content (P450)] were determined. Available data from seven cancer predictors published by others [the Ames test (AMES), mutation in Salmonella typhimurium TA 1537 (TA 1537), structural alerts (SA), mutation in mouse lymphoma cells (MOLY), chromosomal aberrations in Chinese hamster ovary cells (ABS), sister chromatid exchange in hamster ovary cells (SCE), and the ke test (ke)] were also compiled for these 21 chemical carcinogens plus 28 carcinogens and 62 noncarcinogens already published by our laboratory. From the resulting 111 (chemicals) by 11 (individual cancer predictors) data matrix, the five operational characteristics (sensitivity, specificity, positive predictivity, negative predictivity, and concordance) of each of the 11 individual cancer predictors (four biochemical parameters of this study and seven cancer predictors of others) are presented. Two examples of complementarity or synergy of composite cancer predictors were found. To obtain maximum concordance it was necessary to combine both genotoxic and nongenotoxic cancer predictors. The composite cancer predictor (DD or [ODC and P450] or [ODC and ALT]) had higher concordance than did any of the four individual cancer predictors from which it was constructed. Similarly, the composite cancer predictor (TA 1537 or DD or [ODC and P450] or [ODC and ALT]) had higher concordance than any of its five individual constituent cancer predictors. Complementarity or synergy has been demonstrated both 1) among genotoxic cancer predictors (DD and TA 1537) and 2) between nongenotoxic (ODC, P450, and ALT) and genotoxic cancer predictors (TA 1537 and DD).

A problem that hormesis has in being more scientifically accepted is (1) proving that only one mechanism accounts for both the ‘beneficial’ and ‘toxic’ parts of the biphasic dose-response curve and (2) giving substantial evidence against the interpretation that ‘hormesis’ is the sum of many different mechanisms which add up to either ‘beneficial’ or ‘toxic’ in two different parts of the dose-response curve. Hormesis may consist of a initial beneficial dose region where several mechanisms are operating (just for the sake of argument let us say 3 mechanisms) and the overall sum of these 3 mechanisms is ‘beneficial’ to the organism. At higher, toxic, doses, many more mechanisms are operating (just for the sake of argument let us say 8 mechanisms) and the sum of all these 8 mechanisms puts the organism in the ‘toxic’ part of the biphasic dose-response curve.

Organic solvent free procedures are described for the purification of mammalian DNA from rat liver, kidney, spleen, lung and brain. The basis of the purification procedures are the use of the detergent sodium dodecyl sulfate (SDS) and inert polycarbonate filters with 2 mcm pores which retain long macromolecules such as DNA while allowing other cellular constituents to pass through the pores. Neither proteinase K nor use of peristaltic pumps were found to be absolutely required for DNA purification, although it is desirable to use peristaltic pumps to control solution flow rates. Of the 12 different procedures (1.2-4.2 h required) evaluated, purified mammalian DNA contained from 0 detectable to 1.5% of the original protein remaining as a contaminant. With the 4 recommended procedures, the average of 8 experimental values of DNA to protein ratios was 1.81. Depending on the diameter of the individual filter, about 12-90 mcg of rat liver DNA can be purified from each separate tissue or cell sample.

Female Sprague-Dawley rats were given 0, 168, 840, 2550 or 4200 mg/kg of 1,4-dioxane 21 and 4 h before sacrifice. Hepatic DNA damage (by the alkaline elution technique), ornithine decarboxylase activity (ODC), reduced glutathione content, cytochrome P-450 content and serum alanine aminotransferase activity (ALT) were determined. Treatment with 1,4-dioxane increased hepatic DNA damage and cytochrome P-450 content at doses of 2550 and 4200 mg/kg. Large increases in the activity of hepatic ODC were observed at 840, 2550 and 4200 mg/kg of 1,4-dioxane. Thus the data suggest that 1,4-dioxane is a weak genotoxic carcinogen in addition to being a strong promoter of carcinogenesis (a non-genotoxic carcinogen).

Disodium ethylenediamine tetraacetic acid and/or allylisopropylacetamide administration to rat pups did not evoke a premature induction of hepatic δ-aminolevulinic acid synthetase. Administration of iron to adult rats did not alter δ-aminolevulinic acid synthetase activity and had little inductive effect on heme oxygenase activity. Both heme and cobalt/dextran rapidly induced microsomal heme oxygenase by 3–8 fold. Induction of heme oxygenase by heme could be totally blocked by concurrent administration of cycloheximide. These results argue against the hypothesis that iron is the physiological mediator of δ-aminolevulinic acid synthetase activity.

The hepatic biochemical effects of three chlorinated phenols and one phenate were determined. Two oral doses of 1/5 the LD50 of 2,4‐dichlorophenoxyacetic acid, 2,4,5‐trichlorophenol, 2,4,6‐trichlorophenol and 2,3,4,6‐tetrachlorophenol were given 21 and 4 hours before sacrifice to four separate treatment groups of adult female rats. Although 2,4,6‐trichlorophenol is a carcinogen in lifetime rodent studies, no evidence was found for DNA damage in rat liver or white blood cells with the alkaline elution assay. Similarly no compound related changes were found in serum alanine amino‐transferase activity or hepatic glutathione. A dose of 193 mg/kg (slightly over the published rat LD50) of 2,3,4,6‐tetrachlorophenol increased five‐fold the activity of hepatic ornithine decarboxylase. At a dose of 500 mg/kg, 2,4,6‐trichlorophenol increased rat hepatic ODC activity by 106% and cytochrome P‐450 content by 41%. As 2,4,6‐trichlorophenol did not damage DNA in this acute study, it may cause cancer by an epigenetic mecha...

1. 1. The effects of in vivo administration of 6 compounds on the activity of δ-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. 2. 2. The order of decreasing potency in reducing ALA synthetase activity was heme. bilirubin. protoporphyrin IX, bilirubin dimethyl ester, CoCl 2 and FeCl 3 . 3. 3. The chelating agents EDTA and deferoxamine did not prevent heme's repression of ALA synthetase or induction of heme oxygenase activity. 4. 4. The dose response, time course, enzyme subcellular distribution and chelation antagonism studies all suggest that heme itself, and not iron, regulates the rate limiting enzymatic steps of rat hepatic heme synthesis and degradation.

Two oral doses of 1,2-dibromoethane (10-300 mumol/kg) were given to adult female rats 21 and 4 hours before sacrifice. Then hepatic DNA damage, ornithine decarboxylase, cytochrome P-450 content, glutathione content and serum alanine aminotransferase activity assays were performed. In addition, DNA damage was assessed in blood, bone marrow, kidney, spleen and thymus. Of the six organs studied, liver showed the largest amount of DNA damage. Doses at or above 10 mumol/kg EDB caused DNA damage as determined by the alkaline elution technique. Far greater doses (300 mumol/kg, 56.4 mg/kg) of EDB were required to cause other biochemical effects, such as increased activity of ornithine decarboxylase. Thus, the carcinogen EDB caused substantial DNA damage at doses far below those required to show other biochemical effects or frank liver toxicity. DNA damage occurred at a dose level 40-fold lower than that demonstrated in previous studies.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-3H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.

Female Sprague-Dawley CD rats were fed 0, 60, 80, 100, 120 and 140 ppm hexachlorobenzene (HCB) continuously in the diet and 2 successive litters raised. These doses were selected to range from approximately the no observable effect level to lethality in suckling offspring of treated dams. In the F1a generation, the 21-day mortality was 9.2, 19.8, 30.0, 45.4, 93.1 and 92.6% in offspring of dms fed 0, 60, 80, 100, 120 and 140 ppm HCB, respectively. In the F1b generation, a similar mortality of 18.5, 21.5, 19.5, 45, 100, and 94.1% was observed at these 5 dose levels, respectively. The neonatal lethality observed was related to both maternal dose of HCB and the cumulative lactational exposure. Clinical signs of maternal toxicity were not observed and fertility and fecundity were unaffected. In the lungs of HCB treated dams, increased numbers f intraalveolar foamy histiocytes and hypertrophy and proliferation of the lining endothelial cells of pulmonary venules were observed. These microscopic findings of pulmonary effects of HCB confirmed findings of this laboratory.

This paper is inspired by and based upon a field survey of the Carrowmore megalithic cemetery carried out by the writer and Mr Desmond Smith, then Hon. Secretary of the Sligo Field Club, in 1972–73 and revised in 1977 following the clearance of scrub from a number of sites which had hitherto been incapable of proper examination. So far as is known, this is the first attempt at a definitive, if admittedly superficial, survey of this unique assemblage of tombs and other sites since that conducted by Col. W. G. Wood-Martin almost a century ago and recorded in The Rude Stone Monuments of Ireland (1888). The only other definitive survey was that made in 1837 by George Petrie, archaeologist to the Ordnance Survey, and recorded in the Ordnance Survey Letters for County Sligo. In addition, Petrie prepared a map of the area of the cemetery (unfortunately not reproduced in the Ordnance Survey letters) in which he marked and numbered all sites to his knowledge then extant. This map was revised and used by Wood-Martin and it is on this later version that the identification of sites in the present survey is based.A fairly close acquaintance with Carrowmore over the past twenty years or so has encouraged the conviction that the problems posed are of greater variety and complexity than have up to now been acknowledged.

Female Sprague-Dawley rats were given l -dopa during gestation. Several hours after birth, hemorrhages appeared in the interscapular brown adipose tissue of some of the offspring of l -dopa treated rats. Histological examination of the brown fat of these pups showed large hemorrhages and vasodilation. Administration of l -dopa during the first 5 days of gestation and dopamine for the first 7 days of gestation caused a dose-related occurrence of hemorrhage in the offspring. Cross-foster experiments demonstrated that pups exposed to l -dopa in utero had a significantly increased chance of hemorrhage, while pups nursed by l -dopa-treated rats did not. During Days 1–21 of gestation, haloperidol (1 mg/kg/day) partially blocked the effect of l -dopa (100 mg/kg/day) while propranolol (5 mg/kg/day) and phenoxybenzamine (10 mg/kg/day) did not. When given with l -dopa, the decarboxylase inhibitors MK 486 and RO 4–4602 reduced the frequency of hemorrhages. The combination of l -dopa (100 mg/kg), pyrogallol (100 mg/kg), and tranylcypromine (1.5 mg/kg) produced 20.8% hemorrhage in the pups, a significant increase compared to l -dopa (100 mg/kg) alone (7.9%). Among eight different amino acids and amines tested, dopamine was the most hemorrhagic. The dopamine-β-hydroxylase inhibitor, dimethyldithiocarbamate (125 mg/kg), given daily for the first 7 days of gestation, led to brown fat hemorrhage in 17.6% of the offspring, suggesting that dopamine may be implicated in this effect.

To assess possible maternal hepatic and reproductive effects of this uncharged, low molecular weight, lipophilic chlorinated benzene 0, 100, 300 and 1000 mg/kg/day of 1,2,3,4-tetrachlorobenzene (TCB) was orally administered to pregnant rats on days 9–13 of gestation and the animals were killed on day 14 of pregnancy. Phenobarbital and s-naphthoflavone were administered to other pregnant rats as positive hepatic controls. Maternal mortality ( sol7 19 rats ) was increased and body weight gain was greatly decreased in the 1000 mg/kg/day TCB group. Liver to body weight ratio and hepatic microsomal protein content were unaffected by any TCB treatment. On day 14 maternal NADPH-cytochrome c reductase activity was increased at 1000 mg/kg/day, while the maternal hepatic microsomal cytochrome P-450 content was significantly induced by both 300 and 1000 mg/kg/day of TCB. Microsomal N-demethylation of aminopyrine was increased from 2.6 to 4.0 amd 4.5 nmol/mg protein/min at doses of 300 and 1000 mg/kg TCB, respectively. However, maternal hepatic microsomal ethoxyresorufin O-deethylase activity was not consistently increased by TCB. Hepatic glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was increased only by the 1000 mg/kg/day TCB treatment. The rate of microsomal p-nitrophenol and phenolphthalein glucuronidation was increased by TCB administration. Embryonic growth was adversely affected by TCB treatment. Yolk sac diameter, embryonic crown-rump length, and head length were all decreased by treatment with 300 mg/kg/day TCB. This TCB treatment did not significantly elevate the number of dead or abnormal embryos.

Rat-embryo explants removed on day 10.5 of gestation were cultured for 48 hr in various concentrations of HgCl 2 , CH 3 HgCl or glutathione. Dose-related dysmorphogenesis and growth retardation occurred with increasing concentrations of HgCl 2 (1–10 μ m ). Increasing concentrations of CH 3 HgCl (3–100 μ m ) produced a similar pattern of embryonic effects. The rat-serum incubation medium had no detectable level of reduced glutathione ( m ) and only 0.12 m m total sulphydryl groups. Reduced glutathione (60–300 μ m ) added to the incubation medium was relatively non-toxic. The addition of exogenous glutathione to culture medium containing HgCl 2 partially antagonized the embryonic growth retardation and prevented most of the embryolethality observed in cultures to which only HgCl 2 had been added.

In rat liver, allylisopropylacetamide (AIA) treatment strongly induced (25-fold) the activity of rat hepatic ornithine decarboxylase (ODC). By either the oral or the subcutaneous route, AIA produced a long-lasting induction (30 to 40 hours) of hepatic ODC activity. Three analogs of AIA, propylisopropylacetamide (PIA), allobarbital, and allylbenzene, were active ODC inducers while a fourth, allylacetate, was not. Although induction of hepatic aminolevulinic acid (ALA) synthetase activity and the accumulation of hepatic porphyrins depend on the allyl moiety of AIA, this is not the case with hepatic ODC induction. Allylisopropylacetamide did not elevate serum alanine aminotransferase (SGPT) nor did it cause DNA damage, as measured by the alkaline elution assay. Thus, hepatic cell death is not a likely explanation of AIA's long-lasting induction of ODC. As AIA does not belong to any of the common categories of ODC inducers, it may be the chemical prototype of a new class of hepatic ODC inducers.

In order to study the in vitro embryotoxicity and dysmorphogenesis of water insoluble compounds, solvents or chemical delivery systems of low toxicity and teratogenicity to the developing embryo must be found. Therefore, day 10.5 rat embryos were cultured for 2 days in whole rat serum containing 0.1, 0.5 and 2.5 vol.% of ethyl alcohol, dimethylsulfoxide, acetone, Tween 80, corn oil and 10% acetone/90% corn oil. No adverse effects occurred with any of the solvents at the 0.1% concentration level. At 0.5% ethyl alcohol and Tween 80 significantly reduced embryonic growth and increased the incidence of embryonic abnormalities. With the exception of corn oil and acetone/corn oil, embryos cultured in media containing 2.5% of various solvents failed to grow, did not differentiate, and died during the culture period. Corn oil suspended in rat serum by use of ultrasound was non-toxic even at concentrations of 2.5% and 10%. Growth parameters of embryos cultured in serum containing corn oil were indistinguishable from controls and overall morphogenesis was good (particularly at 2.5%). The order of increasing embryotoxicity and dysmorphogenesis of the studied liquids was corn oil less than acetone/corn oil less than dimethylsulfoxide less than ethyl alcohol, acetone less than Tween 80. Any of the 4 water miscible solvents (at 0.1%) or a sonicated suspension of corn oil in serum (up to 2.5%) met the criteria of a non-toxic and non-teratogenic water insoluble compound delivery system for in vitro embryo culture.

Female rats were dosed orally with one-fifth the LD50 of either 1-nitrosopiperidine (a carcinogen), cyclohexylamine, piperidine, 4-carboxy-1-nitrosopiperidine, 4-cyclohexyl-1-nitrosopiperidine or 2,6-dimethyl-1-nitrosopiperidine at 21 and 4 hr before they were killed. The five noncarcinogenic compounds had no effects on any experimental variables examined [hepatic DNA damage, ornithine decarboxylase (ODC) activity, serum alanine aminotransferase (SGPT) activity, cytochrome P-450 and glutathione content]. After administration of 40 mg/kg of 1-nitrosopiperidine, marked hepatic DNA damage and a 3- to 7-fold increase in the activity of hepatic ODC were observed. 1-Nitrosopiperidine (120 mg/kg, 3/5 LD50) caused DNA damage in rat liver and esophagus but not in leukocytes. This higher dose of 1-nitrosopiperidine also increased hepatic ornithine decarboxylase activity by 9-fold. Thus, this hepatic biochemical assay system correctly identified the one carcinogen and the five noncarcinogens in this series of six nitrogen-containing heterocycles.

Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produced characteristic morphological lesions as well as a general inhibition of embryo and yolk sac growth. Increasing concentrations of NADPH in the presence of microsomes and cyclophosphamide produced progressively greater responses. These effects did not occur when microsomes and NADPH were present in the serum medium for the first 2 hours of incubation followed by one washing and then culturing of the conceptuses from hr 2 to hr 48 in a medium containing cyclophosphamide alone. Cytochrome P-450-depleted microsomes did not bioactivate cyclophosphamide to teratogenic or toxic metabolites. The results indicate that cytochrome P-450-dependent microsomal metabolism of cyclophosphamide is required for the embryotoxic and teratogenic effects observed in vitro.

Three chlorinated methanes, carbon tetrachloride, chloroform, and methylene chloride, known to cause liver tumors in rodents, were given by oral gavage to adult female rats both 21 h and 4 h before sacrifice. Then hepatic DNA damage, ornithine decarboxylase (ODC), cytochrome P-450, glutathione content, and serum alanine aminotransferase (SGPT) activity assays were performed. Carbon tetrachloride increased rat hepatic ODC activity and decreased cytochrome P-450 content at doses both below and above cytotoxicity (as measured by increased SGPT activity). At 54 and 160 mg/kg, chloroform increased hepatic ODC activity with minimal or no elevation in SGPT activity. At 480 mg/kg chloroform increased hepatic ODC and SGPT activity. A dose of 1,275 mg/kg methylene chloride caused a small, but significant amount of hepatic DNA damage. When these three compounds are compared on either an equimolar or equitoxic (1/5 LD50) basis, their ability to induce hepatic ODC or increase SGPT activity was carbon tetrachloride greater than chloroform greater than methylene chloride. The results of this biochemical study are interpreted with respect to the ability of chemicals to cause hepatic cancer by either genetic or epigenetic mechanisms.

The level of endogenous root-promoting and inhibiting substances in 3 clones of rhododendron were compared at seasonal intervals in order to study the clonal and seasonal variation in rooting response of cuttings. The highest levels of 4 rooting cofactors in any season were found in both stem and leaf tissue of Rhododendron cv. ‘Cunningham’s White’ followed by ‘English Roseum’. The clone ‘Dr. H. C. Dresselhuys’ contained the least amount of the rooting cofactors. An inhibitor was often found in all clones, but it appeared less responsible for clonal differences in rooting response than variation in levels of the rooting cofactors. Rooting cofactor levels contained in the stem tissue of ‘Cunningham’s White’ were not less than those in the leaf tissue. In contrast, cofactor levels present in the stem tissue of ‘English Roseum’ and ‘Dr. H. C. Dresselhuys’ were less than those in the leaf tissue. The promoting activity of rooting cofactors in all tissues of the clones increased in September and decreased again in November to the level of July extract. The inhibitor found in the July extracts disappeared in September and reappeared in November. Rooting of cuttings of ‘Dr. H. C. Dresselhuys’ was significantly improved by grafting a leaf and bud scion of ‘Cunningham’s White’. On the other hand, scions of ‘Dr. H. C. Dresselhuys’ resulted in decreased rooting of cuttings of ‘Cunningham’s White‘. Rooting capacity of ‘English Roseum’ was less affected by a leaf and bud scion of other clones of Rhododendron.

One spray application of 5000 ppm succinic acid 2, 2-dimethyl hydrazide (Alar) on July 18 or August 22, or 2 spray applications, one on July 18 and another on August 22, significantly retarded new shoot elongation of ‘Collins’ and ‘Bluecrop’ highbush blueberries. The number of flower buds per unit length of new growth was increased. Blossom opening and ripening of the berries was delayed, but fruit size was not appreciably affected. All responses of the 2 cultivars to treatments were more pronounced when they received the 2 spray applications.

1,2,4,5-Tetrachlorobenzene (TCB) is an industrial intermediate used in the production of 2,4,5-trichlorophenoxyacetic acid. This herbicide contains trace quantities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Because of possible maternal hepatic or reproductive effects of this uncharged, low-molecular weight, lipophilic chlorinated benzene 0, 30, 100, 300, and 1000 mg/kg/day of TCB was orally administered to rats on Days 9, 10, 11, 12, and 13 of gestation and the animals were sacrified on Day 14 of pregnancy. No maternal deaths were recorded and body weight gain was significantly decreased only in the 1000 mg/kg/day group. Maternal liver weight, liver to body weight ratio, and hepatic microsomal protein content were unaffected by TCB treatment. Although Day 14 NADPH-cytochrome c reductase activity was not affected, the maternal hepatic microsomal cytochrome P-450 content was significantly increased by administration of 1000 mg/kg/day of TCB. Microsomal N-demethylation of aminopyrine was slightly increased from 2.4 to 3.4 and 3.5 nmole/mg protein/min at doses of 300 and 1000 mg/kg TCB. However, maternal hepatic microsomal ethoxyresorufin O-deethylase activity was greatly increased from 14 to 30, 40, 50, and 49 pmole/mg protein/min in pregnant rats administered 0, 30, 100, 300, and 1000 mg/kg/day TCB. The microsomal rates of p-nitrophenol and phenolphthalein glucuronidation in vitro were not increased by TCB administration. The maternal hepatic microsomal enzyme induction observed after TCB administration to pregnant rats suggests the presence both cytochrome P-450 and P-448 inducers in the sample of 1,2,4,5-tetrachlorobenzene used.

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N -demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo- p -dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[ a ]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[ a ]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.