XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome.

XIST RNA paints and induces silencing of one X chromosome in mammalian female cells, providing a powerful model to investigate long-range chromosomal regulation. This chapter focuses on events downstream from the spread of XIST RNA across the interphase chromosome, to consider how this large noncoding RNA interacts with and silences a whole chromosome. Several lines of evidence are summarized that point to the involvement of repeat sequences in different aspects of the X-inactivation process. Although the "repeat genome" comprises close to half of the human genome, the potential for abundant repeats to contribute to genome regulation has been largely overlooked and may be underestimated. X inactivation has the potential to reveal roles of interspersed and other repeats in the genome. For example, evidence indicates that XIST RNA acts at the architectural level of the whole chromosome to induce formation of a silent core enriched for nongenic and repetitive (Cot-1) DNA, which corresponds to the DAPI-dense Barr body. Expression of repeat RNAs may contribute to chromosome remodeling, and evidence suggests that other types of repeat elements may be involved in escape from X inactivation. Despite great progress in decoding the rest of the genome, we suggest that the repeat genome may contain meaningful but complex language that remains to be better studied and understood.

Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin.

Some 2000 species of cyanobacteria (blue-green algae) occur globally in aquatic habitats. They are able to survive under a wide range of environmental conditions and some produce potent toxins. Toxin production is correlated with periods of rapid growth (blooms) and 25%-70% of blooms may be toxic. Anatoxin-a is an alkaloid neurotoxin that acts as a potent neuro-muscular blocking agent at the nicotinic receptor. Acute toxicity, following consumption of contaminated water, is characterized by rapid onset of paralysis, tremors, convulsions and death. Human exposures may occur from recreational water activities and dietary supplements, but are primarily through drinking water. The current studies were conducted to examine the effect of in utero exposure on postnatal viability, growth and neurodevelopment, to evaluate the potential of in vitro embryotoxicity, and to explore the synergistic relationship between anatoxin-a and the algal toxin microcystin-LR by the oral route. The results of preliminary studies on amphibian toxicity are also reported. Time-pregnant mice received 125 or 200 microg kg(-1) anatoxin-a by intraperitoneal injection on gestation days (GD) 8-12 or 13-17. Pup viability and weight were monitored over a 6-day period. Maternal toxicity (decreased motor activity) was observed at 200 microg kg(-1) in both treatment periods. There were no significant treatment-related effects on pup viability or weight on postnatal day (PND) 1 or 6. The GD 13-17 pups were evaluated on PND 6, 12 and 20 for standard markers of neurodevelopmental maturation (righting reflex, negative geotaxis and hanging grip time). No significant postnatal neurotoxicity was observed. In vitro developmental toxicity was evaluated in GD 8 mouse embryos exposed to 0.1-25 microm anatoxin-a for 26-28 h. Perturbations in mouse yolk sac vasculature were noted from the 1.0 microm concentration in the absence of significant embryonic dysmorphology. Potential algal toxin synergism was tested in mice receiving either 0, 500 or 1,000 microg kg(-1) microcystin-LR by gavage and approximately 50 min later receiving either 0, 500, 1,000 or 2,500 microg kg(-1) anatoxin-a by the same route. No deaths occurred at any dose and no definitive signs of intoxication were observed. Stages 17 and 25 toad embryos (Bufo arenarum) were exposed to 0.03-30.0 mg l(-1) of anatoxin-a for 10 days. Adverse effects included a dose-dependent transient narcosis, edema and loss of equilibrium. Most notable was the occurrence of 100% mortality at the high dose in both groups 6-13 days post-exposure. The observed delay between initial exposure and death is highly unusual for anatoxin-a.

Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

Lack of teratogenicity of microcystin-LR in the mouse and toad.

Microcystin-LR (MC-LR) is a cyanobacterial toxin generated by the organism Microcystis aeruginosa. Although the hepatotoxicity of this chemical has been characterized, the potential developmental toxicity in vertebrates has not been well studied. The purpose of this study was to elucidate the effects of this toxin on the in vivo and in vitro development of mammals and the development of an Anuran (toad). Initial acute toxicity experiments with female CD-1 mice were accomplished with MC-LR administered i.p. in saline. Lethality occurred at 128 and 160 microg kg (-1) and histopathology revealed massive hepatic necrosis with diffuse hemorrhage. Developmental toxicity studies were done with MC-LR administered i.p. for 2-day periods: gestation days 7-8, 9-10 or 11-12. Doses used ranged from 2 to 128 microg kg(-1). On gestation day 17, fetuses were weighed and analyzed for gross morphological and skeletal defects. No treatment-related differences were seen in litter size, viability, weight or the incidence of anomalies. Groups of dams dosed with 32-128 microg kg(-1) on gestation days 7-8, 9-10 or 11-12 were allowed to give birth and the growth and development of their pups were followed postnatally. There were no significant effects noted in the offspring of the treated dams. Neurulation-staged CD-1 mouse conceptuses were exposed to 50-1000 nM MC-LR in whole embryo culture for 24 h. No significant increase in abnormalities or developmental delays was observed. Finally, exposure of the developing toad. Bufo arenarum was done from stage 17 (tail bud) for 10 days at concentrations of 1-20 mg l(-1). No effect on morphological development or survival was noted in any exposed groups. These data indicate that microcystin does not appear to affect development adversely in the mouse (in vivo or in vitro) or the toad at the doses and exposure parameters used.

Ectopic XIST transcripts in human somatic cells show variable expression and localization.

XIST encodes a functional RNA that is expressed exclusively from the inactive X in female mammals and is required for the silencing of most of the genes on the chromosome. XIST transcripts remain in the nucleus, and their specific localization to the inactive X is important for silencing; however, it is not known how these transcripts localize to the inactive X chromosome. Expression of mouse and human XIST from ectopic sites has suggested that localization to the chromosome from which the gene is expressed may be dependent upon either the copy number of the integrated constructs or the level of ectopic XIST expression. To further examine the behavior of XIST transgenes when expressed from ectopic sites, we introduced an XIST-containing PAC into the human male somatic cell line HT-1080. In five different transformant clones, the degree of localization and associated DNA condensation of the surrounding chromatin varied within nuclei of the same clone, as well as among different clones. Comparing the number of integrated transgenes and the levels of XIST expression revealed that neither factor was sufficient for a tight localization of the XIST signal. Therefore, the extent of expression and localization of XIST transcripts from ectopic transgenes is likely dependent upon many interacting factors, including the number of integrated transgenes, the level of XIST expression, and the site of integration.

Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine.

The programmed cell death of the stratified squamous epithelial cells comprising human epidermis culminates in abrupt transition of viable granular keratinocytes (KC) into dead corneocytes sloughed by the skin. The granular cell-corneocyte transition is associated with a loss in volume and dry cell weight but the mechanism for and biological significance of this form of keratinocyte apoptosis remain obscure. We show that terminally differentiated KC extrude into the intercellular spaces of living epidermis the cytoplasmic buds containing randomly congregated components of the cytosol as well as filaggrin, a precursor of the natural moisturizing factor. The discharge of secretory product is reminiscent of holocrine secretion, suggesting the term 'apoptotic secretion' for this novel, essential step in the process of cornification. The secretory product may become a part of the glycocalyx (a.k.a. 'intercellular cement substance' of epidermis) and serve as a humectant that counterbalances the osmotic pressure imposed by the natural moisturizing factor located in the stratum corneum comprised by corneocytes. The apoptotic secretion commences upon secretagouge action of acetylcholine which is synthesized and released by KC. A combination of a cholinergic nicotinic agonist and a muscarinic antagonist which increases intracellular calcium levels is required to trigger the apoptotic secretion. Analysis of the relative amounts of cholinergic enzymes and receptors expressed by KC capable of secretion and the pharmacological profiles of secretion regulation revealed an upward concentration gradient of free acetylcholine in epidermis which may provide for its unopposed secretagogue action via the m1 muscarinic and the alpha7, and alpha9 nicotinic receptor types expressed by KC at the latest stage of their development in the epidermis.

Nonviral gene delivery to the lateral ventricles in rat brain: initial evidence for widespread distribution and expression in the central nervous system.

The use of DNA for nonviral gene expression depends on several factors. These include (i) delivery and accessibility to the targeted tissue, (ii) protection from extracellular degradation, (iii) sufficient uptake by cells of interest, and (iv) protection from intracellular degradation to allow translation of adequate levels of intracellular or secreted proteins. As an initial step in demonstrating the feasibility of nonviral, cationic lipid-mediated gene therapy, we present evidence for the successful delivery and expression of heat shock protein Hsp70 and reporter gene enzymes in the central nervous system (CNS) of the rat after injection into the lateral ventricle. Gene delivery is accomplished using optimized formulations of plasmid DNA, which have been complexed with the cationic lipid MLRI. Results from DNA vectors encoding for green fluorescent protein (GFP), luciferase, and Hsp70 are reported. Standard immunofluorescent methods were used to demonstrate widespread expression of the reporter proteins and of Hsp70. Stereology analysis has been completed on three coronal sections, which illustrates the distribution of expression along the longitudinal axis. These initial findings support the further development of nonviral, lipid-mediated gene delivery technology for transient expression of protective, intracellular proteins and represent an important step leading to in vivo studies to identify potential clinical benefits.

Recovery and serious brain disorders: the central role of families in nurturing roots and wings.

Recovery in severe mental illness is a powerful concept for consumers, invoking a journey through understanding and acceptance of illness and disability, along with hope for a quality life, self-empowerment, and responsibility. Although not widely recognized, the journey toward recovery is also important for family members of individuals with severe mental illnesses, who often serve in a care-giving or supportive role for their loved ones. From the family perspective, to make recovery a real possibility, several issues must be confronted. First, progress toward recovery must acknowledge and involve care-giving and supportive family members, recognizing their significant role in the lives of many individuals with severe mental illnesses as well as the journey of family members in understanding and accepting a severe mental illness in a loved one. Second, a real, recovery-oriented system implements treatments and supports shown to be effective. In this day and age, the gap between research findings and ordinary care condemns too many consumers and their families to outcomes far short of what is possible. This includes not only medications, psychotherapy, employment services, dual diagnosis services, housing and other supports for consumers, but also family education, shown to be effective by a large body of research. Third, a recovery-oriented system of care cannot afford to dance around the thorniest and most controversial issues in severe mental illnesses-such as individuals with the most intractable forms of illness or the relatively infrequent but very real situations in which either consumers or family members are assaultive or abusive. All of these elements are essential if recovery is to be more than a slogan, but rather a true goal in a system of care that respects consumers with these disorders and their care-giving family members.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Inactive matrix metalloproteinase 2 is a normal constituent of human glomerular basement membrane. An immuno-electron microscopic study.

Remodelling of the extracellular matrix requires tight control not only of matrix synthesis, but also of matrix degradation. Control of matrix degradation is achieved mainly through the matrix metalloproteinase (MMP) enzymes. In the glomerulus, MMP-2 and MMP-9 are believed to be particularly important, as they have activity against type IV collagen. This study has demonstrated by immuno-electron microscopy that most of the immunoreactivity for MMP-2 in the normal glomerulus is located within the glomerular basement membranes and mesangial matrix. mRNA for MMP-2 is also detectable in normal glomeruli, but the other main gelatinase, MMP-9, could not be localized by immuno-electron microscopy. In the normal glomerulus, it seemed likely that MMP-2 is present in an inactive form. To confirm this, in situ zymography was carried out using frozen sections of normal kidney. Baseline activity of normal kidney was relatively weak, but this was dramatically increased by chemical activation of metalloproteinases. The results imply that MMP-2, in an inactive form, is a normal constituent of the extracellular matrix and glomerular basement membranes. Activation would presumably render the matrix 'self-degrading'; membrane-bound MMPs (MT-MMPs) seem particularly likely to be involved in leukocyte penetration of basement membranes in inflammation.

Medication discontinuation and symptom provocation in research: a consumer and family perspective.

Research involving symptom provocation and medication discontinuation, although common throughout medicine, raises some significant issues for consumers and family members facing severe mental illnesses. Reasons for our attention to these research approaches include the apparent prevalence of this research; the prospect of significant distress, illness, or disability, especially in the event of medication discontinuation; questions about the scientific strength of at least some challenge studies; the changing nature and pressures on research, including its "deinstitutionalization," and the increasing role of private funders; early evidence of consumer concerns about medication discontinuation; the possibility of long-term damage as a result of delayed treatment; concerns about subject recruitment methods and the adequacy of informed consent for these studies; and the lack of data concerning consumer experience and outcome from this research. Because research involving symptom provocation and medication discontinuation can present significant risks to the consumer participating in the study, we must take steps to make sure of the following: that the use of this research methodology be minimized as much as possible; that it meets the very highest standards of scientific merit and necessity; that it includes the strongest possible protections for human subjects, including optimal informed consent about the nature and risks of the study and communication with care-giving family members and others; that in discontinuation studies research protocols precisely spell out in advance the clinical situations that will trigger intervention and what the intervention will be, and furthermore, that discontinuation studies should assure adequate clinical monitoring for early identification and treatment of signs of clinical distress or deterioration; and that researchers should begin systematically collecting and publishing information on the outcomes for and experiences of consumers in these types of studies. Enhanced oversight of these research protocols, by funders and IRBs is essential. Finally, our consideration of these issues has raised concerns for us about recruitment methods, which warrant examination; and we would like to see attention to placebo control use, insofar as it is necessary to show new medications are effective.

Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation.

The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate. Transcriptional activation of algD results in the high-level synthesis of alginate, an important P. aeruginosa virulence factor with antiphagocytic and adherence properties. Previously, we have identified a protein(s), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription. In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution of a protein preparation from an SDS-PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays. A biochemical enrichment procedure, resulting in a 130-fold enrichment for AlgZ, was devised, the protein identified and a partial amino-terminal sequence obtained. Using the P. aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli. Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P. aeruginosa. A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon-helix-helix family of DNA-binding proteins. An algZ deletion mutant was constructed in the mucoid CF isolate FRD1. The resulting strain was non-mucoid and exhibited no detectable algD transcription. As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT. The cloning of algZ is a crucial step in determining the mechanism of algD activation.

Glomerular expression of nephrin is decreased in acquired human nephrotic syndrome.

BACKGROUND: Nephrin recently has been identified as a putative adhesion molecule, expressed in the glomerulus, in which mutations cause congenital nephrotic syndrome of Finnish type. We sought to determine whether expression of nephrin is altered in human glomeruli in patients with acquired nephrotic syndrome. METHODS: We performed PCR amplification of nephrin cDNA, using cDNA previously prepared from single human glomeruli plucked fresh from the surface of human renal biopsies. We had available four cases of nephrotic syndrome (one membranous, three minimal change) and six normal controls. PCR product quantitation was by gel densitometry, confirmed by enzyme-linked immunosorbent assay using a specific oligonucleotide probe. Results were corrected for reaction efficiency and glomerular cellularity by expression as a ratio to levels of the 'housekeeping gene' glyceraldehyde phosphate dehydrogenase. RESULTS: Glomerular levels ofnephrin mRNA are significantly decreased in cases of minimal change nephrotic syndrome. An apparent reduction was also seen in the single case of membranous nephropathy which was available for study. CONCLUSIONS: Abnormalities of nephrin expression appear to be associated with acquired as well as congenital causes of human nephrotic syndrome.

Percutaneous absorption and disposition of [14C]chlordecone in young and adult female rats.

The objective of this study was to evaluate the effect of age and dosage on percutaneous absorption and disposition of [14C]chlordecone (Kepone) and to describe results using a physiological based pharmacokinetic (PBPK) model. Female Fischer 344 rats 33 and 82 days old were used as the young and adult animal models, respectively, and were studied over a 10-fold dose range. [14C]Chlordecone (0.286 micromol/cm2) was applied to dorsal skin (2. 3% BSA) and radioactivity was quantified in selected tissues and excreta up to 120 h. Absorption and disposition were also determined at three dose levels up to 2.68 micromol/cm2; fraction absorbed decreased as dose increased. In vitro percutaneous absorption was measured by static and flow-through methods; these yielded similar penetration rates, which were lower than those obtained in vivo. In vivo percutaneous absorption over 120 h was 14.4+/-0.99 and 14.2+/-1. 5% dose in young and adults, respectively. Organ and tissue content increased over time (carcass>liver>kidney), indicating prolonged absorption. Statistical differences between young and old were found for liver, skin, and urine, but not for absorption. Excretion occurred primarily in feces, but also in urine. A biophysically based percutaneous model was fitted to both young and adult in vivo absorption data. This was embedded in a whole body PBPK model which, upon optimization with SAAM II, estimated apparent tissue partition coefficients, urinary and fecal excretion rates, and parameters characterizing hepatic nonlinear uptake of bound chlordecone. The model reasonably predicted tissue chlordecone content at higher doses, when decreased absorption was accounted for.

Reproducibility in the quantification of mRNA levels by RT-PCR-ELISA and RT competitive-PCR-ELISA.

The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene from isolated renal glomeruli. We used an enzyme-linked immunosorbent assay (ELISA) to quantify PCR products. We also report our PCR-based method for constructing a competitor DNA identifiable independently of the native product. Our results show that the entire RT-PCR and ELISA process had a standard deviation (SD) of less than 10% (n = 10). This compared to an SD of less than 13% (n = 10) in PCR and ELISA. The SD for ELISA alone was less than 11% (n = 10). RT-cPCR quantitation gave an SD of approximately 15% (n = 10). These results support the use of standard RT-PCR for the relative quantitation of mRNA. RT-cPCR is also suited to relative quantitation, but it is also independent of the amplification saturation curve and permits the identification of differences in cellularity between samples.

In vitro methylation of inorganic arsenic in mouse intestinal cecum.

The capacity of mouse intestinal cecal microflora to methylate inorganic arsenicals (iAs) was examined in vitro under conditions of restricted bacterial growth. Cecal contents incubated under anaerobic conditions at 37 degrees C for 21 hr methylated up to 40% of either 0.1 microM arsenite (iAsIII) or 0.1 microM arsenate (iAsV). Methylarsenic (MAs) was the predominant metabolite; however, about 3% of either substrate was converted to dimethylarsenic (DMAs). Over the first 6 hr, the rate of methylation was several times greater for iAsIII than for iAsV. There was a 3-hr delay in the production of methylated metabolites from iAsV, suggesting that reduction of iAsV to iAsIII before methylation could be rate limiting. Over the concentration range of 0.1 to 10 microM of iAsIII or iAsV, there was an approximately linear increase in the production of MAs and DMAs. There was evidence of saturation or inhibition of methylation at 100 microM of either substrate. Substrate concentration had little effect on MAs/DMAs ratio. Incubation of cecal contents at 0 degrees C abolished methylation of either arsenical. Under aerobic or anaerobic conditions, cecal tissue homogenates produced little MAs or DMAs from either arsenical. Addition of potential methyl group donors, L-methionine and methylcobalamin, into cecal contents significantly increased the rate of methylation, especially for iAsV. Addition of glutathione, but not L-cysteine, had a similar effect. Selenite, a recognized inhibitor of iAs methylation in mammalian tissues, inhibited methylation of either substrate by cecal contents. These data suggest that cecal microflora are a high capacity methylation system that might contribute significantly to methylation of iAs in intact animals.

In vivo disposition of p-substituted phenols in the young rat after intraperitoneal and dermal administration.

The objective of this study was to examine the 120-hr disposition of phenol and four p-substituted congeners after ip and dermal administration in the 29-day-old female rat. The dermal absorption was very high (66-80% of the dose) for phenol, cyanophenol, heptyloxyphenol and nitrophenol, but minimal for hydroxybenzoic acid (2%). The major portion of the dose for all of the phenols not absorbed dermally in 24 hr was washed from the skin. Only minor amounts (1-2%) were detected in the treated skin at 120 hr. Urinary excretion was the predominant means of elimination for these phenols and occurred primarily within 24 hr after dermal and ip administration. However, the excretion of heptytoxyphenol after administration by both routes differed from that of the other compounds, with more of it detected in the faeces. The profile of metabolites in urine (collected at 12-24 hr) from the animals dermally treated with phenol, cyanophenol, heptyloxyphenol and nitrophenol showed only peaks that eluted earlier than the parent compound, which suggests that conjugates or more polar metabolites were formed and excreted. The difference in dermal absorption between hydroxybenzoic acid and the other phenols may be due to potential ionization of the p-substituted carboxylic acid group of hydroxybenzoic acid. This suggests that, at least for the phenols examined in this study, physicochemical characteristics other than just lipophilicity can affect in vivo dermal absorption.