Characterizing dye bias in microarray experiments.

MOTIVATION: Spot intensity serves as a proxy for gene expression in dual-label microarray experiments. Dye bias is defined as an intensity difference between samples labeled with different dyes attributable to the dyes instead of the gene expression in the samples. Dye bias that is not removed by array normalization can introduce bias into comparisons between samples of interest. But if the bias is consistent across samples for the same gene, it can be corrected by proper experimental design and analysis. If the dye bias is not consistent across samples for the same gene, but is different for different samples, then removing the bias becomes more problematic, perhaps indicating a technical limitation to the ability of fluorescent signals to accurately represent gene expression. Thus, it is important to characterize dye bias to determine: (1) whether it will be removed for all genes by array normalization, (2) whether it will not be removed by normalization but can be removed by proper experimental design and analysis and (3) whether dye bias correction is more problematic than either of these and is not easily removable. RESULTS: We analyzed two large (each >27 arrays) tissue culture experiments with extensive dye swap arrays to better characterize dye bias. Indirect, amino-allyl labeling was used in both experiments. We found that post-normalization dye bias that is consistent across samples does appear to exist for many genes, and that controlling and correcting for this type of dye bias in design and analysis is advisable. The extent of this type of dye bias remained unchanged under a wide range of normalization methods (median-centering, various loess normalizations) and statistical analysis techniques (parametric, rank based, permutation based, etc.). We also found dye bias related to the individual samples for a much smaller subset of genes. But these sample-specific dye biases appeared to have minimal impact on estimated gene-expression differences between the cell lines.

Acute, distribution, and subchronic toxicological studies of succinate tartrates.

The estimated single-dose oral toxicity (50% lethality) of succinate tartrates (ST) was 2-3 g/kg in rats. ST produced minimal to moderate dermal irritation but no evidence of systemic toxicity in a standard acute percutaneous toxicity test in rabbits. ST was not an eye irritant in a standard rabbit low-volume eye irritation test. ST was not genotoxic in a series of six genotoxicity tests. A 14-day oral gavage study in rats at a dose range of 0.05-1.0 g ST/kg/day produced only gastric irritation. The no-observed-effect level (NOEL) for gastric irritation was 0.1 g/kg for males and 0.05 g/kg for females. A 28-day percutaneous toxicity study in rabbits produced minimal to moderate dermal irritation and no adverse systemic effects at a high dose of 450 mg ST/kg/day. Single-dose absorption, distribution, and elimination (ADE) studies in male rats showed that 10-15% of an oral dose and 1-3% of a dermal dose were absorbed. Approximately 98% of the orally administered ST was eliminated as 14C in urine, feces, or expired CO2 after 72 hr. Approximately 80% of the dermally absorbed 14C dose was eliminated in urine, feces, or expired CO2 after 72 hr. In conclusion, no adverse effects were noted in acute toxicity, genotoxicity, or subchronic toxicity studies conducted with ST.

Lemon aesthetics in hand dishwashing detergents do not influence reported accidental ingestion frequency and volume.

Accidental ingestion reports for 2 nationally distributed hand dishwashing detergents with similar formulations were compared to determine the possible effect of lemon aesthetics on ingestion frequency and volume. Data were compared over almost a 2-year period (May 1986 to April 1988), and a total of 2141 accidental ingestion reports for the 2 products were reviewed. There was no difference in the reported ingestion volume of lemon-scented versus nonlemon-scented product. Fewer accidental ingestion reports were received for lemon-scented than for nonlemon-scented products, even when data were normalized for market shipments of each product.

Subchronic oral toxicity of succinate tartrates in rats.

A subchronic oral toxicity study was carried out in rats. Groups of ten male and ten female Fischer 344 rats were administered succinate tartrates (ST) at concentrations of 0 (control), 0.01, 0.05, 0.1 and 0.5%, w/w, active ingredient in the drinking-water for 91 days. No mortality was caused by the treatment and no toxicological signs were observed. No treatment-related effects were noted in wk 13 body weights, body-weight gains or food consumption. Urinary haematological and clinical chemistry parameters showed no treatment-related effects, with the exception of a significant decrease in serum magnesium levels in all ST-treated females, but not including the 0.1% dose group. At necropsy, no treatment-related changes in absolute or relative organ weights were noted with the exception of the adrenals. A significant increase in absolute and relative adrenal weights was observed in females administered 0.1% and 0.5% ST; however, the overall analysis of variance was significant only for the relative adrenal weights. In addition, no treatment-related gross or microscopic tissue changes were observed in any organs or tissues including the adrenals. In the absence of histological changes, the adrenal weight changes were not considered biologically significant. The no-observed-adverse-effect level for this study was thus 0.5% ST.

Evaluation of the developmental toxicity of succinate tartrates in rats.

The potential for succinate tartrates (ST) to induce developmental toxicity in Sprague-Dawley CD rats has been evaluated. ST dose levels of 250, 500 and 1000 mg/kg body weight/day were administered in the drinking-water on days 6-15 of gestation. Control animals received distilled water. Caesarean sections were performed on gestation day 20 and the foetuses were removed for teratological evaluation. No significant maternal or developmental toxicity was observed at any dose level. Based on these observations, the no-observed-effect level for ST developmental toxicity is greater than or equal to 1 g/kg/day, which was the highest dose tested.

Profile of accidental ingestion calls received via a toll-free line on detergent product labels.

Accidental ingestion comments received from May 1986 to April 1988 via a toll-free telephone line on detergent product labels were profiled to learn the weight of ingesters, the reported ingestion volumes, and the symptoms reported for several different detergent product categories. Most of the ingesters weighed 20 kg or less; however, 10% of the ingesters weighed 70 kg or more. Most detergent product ingestions involved 1 teaspoon of product or less. Reported symptoms varied with product type and ingestion volume.

Subchronic percutaneous toxicity testing of two liquid hand dishwashing detergents.

Subchronic percutaneous toxicity studies were conducted on two liquid dishwashing detergents containing anionic surfactants (C12-14 alkylethoxylate sulphate) to assess the safety of these materials for human exposure. The detergents were administered dermally to the shaved backs of rabbits (dose volume of 2 ml/kg body weight) at concentrations of 0, 0.5, 1.0 and 2.5% in distilled water for 91 days. No adverse systemic effects were demonstrated by assessment of haematological parameters or by gross or microscopic tissue examination. Transient slight to moderate dermal irritation at the detergent application site was observed with detergent A. Slight to moderate dermal irritation confined to the detergent application site was noted in the detergent B study.

Hepatic effects of acrylamide in rainbow trout.

Acrylamide effects on hepatic mixed function oxidase activity were examined in rainbow trout exposed to waterborne acrylamide monomer (0-50 mg/liter) for 14 days. Acrylamide doses of 25 mg/liter and higher produced reversible dose-related histological lesions in the liver characterized by necrosis around the central vein. A profound selective decrease in ethoxyresorufin-O-deethylase activity was observed in hepatic microsomes from acrylamide-treated trout. However, in vitro preincubation of acrylamide with untreated rainbow trout hepatic microsomes with or without NADPH did not produce a similar decrease in ethoxyresorufin-O-deethylase activity. Structural analysis of a trout biliary metabolite of acrylamide, although not identified, revealed the presence of primary alcohol, carboxylic acid, and amide carbonyl functional groups.

Behavioral and histological effects of acrylamide in rainbow trout.

A histological and behavioral study was used to assess whether acrylamide produced neurotoxic effects in rainbow trout. Swimming performance of trout exposed to 0, 12.5, or 25 mg/liter acrylamide for 15 days was unaffected. Swimming performance of animals exposed to 50 mg/liter acrylamide for a similar time period was compromised by morbidity and mortality of the animals in this treatment group. The absence of dose-related histological lesions in central neurons, peripheral neurons or muscle suggested that the observed deficit in swimming performance was due to a generalized toxic response. Acrylamide treatment produced dose-related lesions in the gill and liver of rainbow trout.

Uptake, disposition, and elimination of acrylamide in rainbow trout.

The uptake, disposition, and elimination of [2,3-14C]acrylamide was studied in fingerling rainbow trout exposed to 0.388 and 0.710 mg/liter [2,3-14C]acrylamide at 12 degrees C under static water conditions for 72 hr. 14C in carcass and viscera was determined at times ranging from 4 to 72 hr after the beginning of the exposure period and 4 to 96 hr after transfer of the fish to fresh flowing water for the elimination studies. Uptake of 14C was initially rapid and plateaued after 72 hr of acrylamide exposure. No appreciable bioaccumulation occurred in carcass or viscera at either exposure concentration and 14C distributed approximately equally to all tissues studied. Elimination of 14C from carcass and viscera was biphasic with a terminal half-life of approximately 7 days. 14C elimination was not uniform in all tissues studied with the most rapid elimination occurring in blood and gill and the slowest elimination occurring in muscle and intestine. In addition, 10 to 15% of the initial total 14C in carcass or viscera was nonextractable and was associated with the protein fraction of the sample at all time points in the depuration period. Approximately 20% of an ip administered dose of [14C]acrylamide was eliminated via the gills, 7% via the urine, and less than 1% via the bile in 2 hr. At least three biliary metabolites were isolated by HPLC.

Differential tolerance to the intestinal inhibitory effect of opiates in mice.

The occurrence of unidirectional noncross-tolerance to heroin and etorphine induced antinociception in morphine tolerant mice prompted this study to determine whether this phenomenon occurs for opiate induced inhibition of intestinal transit. Tolerance to the intestinal inhibitory effect (charcoal meal test) of s.c. morphine, etorphine and heroin was reflected by an increase in the ED50 value and in most cases, a flattening of the dose response curve in morphine and etorphine pelleted mice (tested with pellet in place). A similar response was noted following intracerebroventricular administration of these agents in morphine pelleted mice. More tolerance developed to the relatively hydrophilic compound morphine than to the lipophilic compounds etorphine and heroin. Factors related to hydrophilicity and lipophilicity may be involved in the development of tolerance to opiates in the intestine although unidirectional non cross-tolerance did not occur.