Dean W. Roberts

Quantitative Determination of Skin Penetration of PEG-Coated CdSe Quantum Dots in Dermabraded but not Intact SKH-1 Hairless Mouse Skin

Many cosmetics, sunscreens, and other consumer products are reported to contain nanoscale materials. The possible transdermal absorption of nanoscale materials and the long-term consequences of the absorption have not been determined. We used polyethylene glycol coated cadmium selenide (CdSe) core quantum dots (QD; 37 nm diameter) to evaluate the penetration of nanoscale material into intact, tape stripped, acetone treated, or dermabraded mouse skin. QD were suspended in an oil-in-water emulsion (approximately 9 microM) and the emulsion was applied at 2 mg/cm(2) to mouse dorsal skin pretreated as follows: intact; tape stripped to remove the stratum corneum; acetone pretreated; dermabraded to remove stratum corneum and epidermis. QD penetration into the skin was monitored in sentinel organs (liver and regional draining lymph nodes) using inductively coupled plasma mass spectrometry analysis of cadmium (from the CdSe QD). No consistent cadmium elevation was detected in the sentinel organs of mice with intact, acetone pretreated, or tape-stripped skin at 24- and 48-h post-QD application; however, in dermabraded mice, cadmium elevations were detected in the lymph nodes and liver. QD accumulation (as cadmium) in the liver was approximately 2.0% of the applied dose. The passing of QD through the dermabraded skin was confirmed using confocal fluorescence microscopy. These results suggest that transdermal absorption of nanoscale materials depends on skin barrier quality, and that the lack of an epidermis provided access to QD penetration. Future dermal risk assessments of nanoscale materials should consider key barrier aspects of skin and its overall physiologic integrity.

The effect of propylene glycol on the P450-dependent metabolism of acetaminophen and other chemicals in subcellular fractions of mouse liver

Propylene glycol (PG) decreases the hepatotoxicity of acetaminophen (APAP). To elucidate the mechanism for this response, we measured the effect of PG on the in vitro metabolism of APAP by subcellular liver fractions from 6-10 week-old male B6C3F1 mice. The fractions were assayed for their ability to bioactivate APAP to N-acetyl-p-benzoquinone imine, which was trapped as APAP-glutathione conjugates or APAP-protein adducts, and for dimethyl-nitrosamine-N-demethylase (DMN), 4-nitrophenol hydroxylase (4-NPOH), and phenacetin-O-deethylase (PAD) activities. Activity in the crude mitochondrial-rich (10,000 x g pellet) fraction was low and PG had no effect. PG inhibited DMN and 4-NPOH, indicators of IIE1-dependent activity, and the formation of APAP-glutathione conjugates and APAP-protein adducts in both heavy (15,000 x g pellet) and light (100,000 x g pellet) microsomes. PAD, a measure of IA2-dependent activity, was not inhibited. These data demonstrate that PG selectively inhibits IIE1 activity, including the bioactivation of APAP, and implicates this as the mechanism for PG-mediated protection of APAP hepatotoxicity in mice.

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme‐catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki′ of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki′ of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.

Effects of lithium and rubidium on immune responses of rats.

Rats were treated with LiCl or RbCl in drinking water for 65 days. Spleen cells from both treated groups exhibited significantly greater proliferative responses to lipopolysaccharide (LPS) than those from untreated controls. Responses to concanavalin A (Con A) were not affected. Cytotoxic activities of natural killer (NK) cells from both treated groups were significantly less than those from untreated controls. In vitro, Li augmented responses of spleen cells to LPS, but the same doses of Rb suppressed the responses. Effects on responses to Con A were variable. Both Li and Rb alone had a small mitogenic effect on spleen cells.

Effects of the Adulteration Technique on the Near-Infrared Detection of Melamine in Milk Powder

The United States Pharmacopeial Convention has led an international collaborative project to develop a toolbox of screening methods and reference standards for the detection of milk powder adulteration. During the development of adulterated milk powder reference standards, blending methods used to combine melamine and milk had unanticipated strong effects on the near-infrared (NIR) spectrum of melamine. The prominent absorbance band at 1468 nm of melamine was retained when it was dry-blended with skim milk powder but disappeared in wet-blended mixtures, where spray-dried milk powder samples were prepared from solution. Analyses using polarized light microscopy, Raman spectroscopy, dielectric relaxation spectroscopy, X-ray diffraction, and mass spectrometry indicated that wet blending promoted reversible and early Maillard reactions with lactose that are responsible for differences in melamine NIR spectra between wet- and dry-blended samples. Targeted detection estimates based solely on dry-blended reference standards are likely to overestimate NIR detection capabilities in wet-blended samples as a result of previously overlooked matrix effects arising from changes in melamine hydrogen-bonding status, covalent complexation with lactose, and the lower but more homogeneous melamine local concentration distribution produced in wet-blended samples. Techniques used to incorporate potential adulterants can determine the suitability of milk reference standards for use with rapid detection methods.

Behaviorally conditioned suppression of murine T-cell dependent but not T-cell independent antibody responses

The aversive and immunosuppressive effects of cyclophosphamide (CY, 250 mg/kg IP), an unconditioned stimulus (UCS), were paired with the presentation of a novel saccharine flavored drinking solution (SAC), a conditioned stimulus (CS), in female Balb/c mice. The objective was to determine the temporal relationship between presentation of the CS (SAC) and immunization with sheep red blood cell (SRBCs), a T-cell dependent antigen, and type III pneumococcal polysaccharide (S3), a T-cell independent antigen, on subsequent antibody responses. Reexposure to the CS or UCS occurred on days -4, -2, 0, +2, or +4 relative to immunization. Primary antibody responses in each group were measured six days following immunization. A strong association between the CS and the UCS developed, producing flavor aversions as evidenced by decreased SAC consumption. CY administration by itself consistently suppressed both types of antibody responses. CS presentation (i.e., SAC) had no significant effect on anti-S3 antibody response. However, the anti-SRBC response was significantly depressed following CS exposure. Exposure to the CS only on days -4 or +2 relative to immunization resulted in statistically significant suppression of antibody response to SRBCs while exposure on days -2, 0, and +4 resulted in anti-SRBC antibody suppression that did not reach significance. These results support the hypothesis that conditioning of antibody responses is relatively specific for T-cell dependent antigens, and that the timing of CS presentation relative to immunization is important in conditioning a suppression of antibody responses.

Evaluation of the immune response to type III pneumococcal polysaccharide as a means to evaluate T-independent immune function in the rat.

Since the rat is frequently the experimental animal of choice for toxicology testing, studies were undertaken to adapt assays routinely used to evaluate immune function in mice so that immune function could likewise be evaluated in collaborative projects employing toxicant-treated rats. Contrary to previous reports in the literature, Type III pneumococcal polysaccharide (S3) was immunogenic in rats. Specific antibody responses to S3 were demonstrated in two strains of rats following immunization by either the subcutaneous (sc) or intraperitoneal (ip) route with purified S3, with S3 contained in polyvalent pneumococcal polysaccharide vaccine (pneumovax), or with heat-killed Type III Streptococcus pneumoniae. Dose-response studies demonstrated that the optimal immunizing dose in Sprague-Dawley or Fischer rats was 25 micrograms S3. Reimmunization with S3 on d 21 did not produce an anamnestic response, and the kinetic data were consistent with S3 being a thymus-independent (T-independent) antigen in the rat. In contrast to our previous studies in the mouse, concurrent sc or ip injections of pertussis vaccine did not modify the response to S3 in rats. Sprague-Dawley rats acquired the capacity to respond immunologically to S3 between 24 and 31 d of age. In mature animals, sex had no effect on the ability to respond to S3. The utility of this model as a means of characterize toxicant-induced immune dysfunction was demonstrated using the prototype immunotoxicant cyclo-phosphamide.

Acetaminophen-induced alterations in pancreatic β cells and serum insulin concentrations in B6C3F1 mice

Administration of acetaminophen (500 mg/kg) to male B6C3F1 mice resulted in alterations of pancreatic beta cell ultrastructure. These alterations were characterized by pronounced intercellular spaces, cytoplasmic vacuolization, damaged membranes of cytoplasm, secretory granules, and other organelles, and pyknotic nuclei with disrupted membranes. Concomitant with these changes, acetaminophen also caused increaes in serum insulin concentrations from 24 microU/ml at 0 time to 160 microU/ml at 8 hr and increases in serum alanine aminotransferase (ALT) concentrations from 42 to 13,279 U/liter, which indicated hepatic damage. Quantitation of 3-(cystein-S-yl)acetaminophen adducts in hepatic 10,000g supernatant protein using a particle concentration fluorescence immunochemical assay indicated a positive correlation between binding and the occurrence of the hepatotoxicity consistent with what has been previously reported; however, 3-(cystein-S-yl)acetaminophen protein adducts were not detected in pancreatic 10,000g supernatant. Immunohistochemical analysis of the liver and pancreas from acetaminophen-treated mice revealed acetaminophen-protein adducts in the centrilobular regions of the liver but not in the pancreatic islets. Doses of 100 and 200 mg/kg produced no evidence of hepatotoxicity and no increase in serum insulin; 300 mg/kg and higher doses produced both hepatotoxicity and increased serum insulin concentrations. A comparison of the time course for the increase in serum levels of ALT and insulin following a toxic dose of acetaminophen indicated that the increase in ALT preceded the increase in insulin. Thus the hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts in liver, which supports the concept that this toxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the toxicity of acetaminophen to beta cells in the pancreas is apparently not mediated by this mechanism.

Development of an Avidin-Biotin Amplified Enzyme-Linked Immunoassay for Detection of DNA Adducts of the Human Bladder Carcinogen 4-Aminobiphenyl

4-Aminobiphenyl (ABP) is a known human urinary bladder carcinogen which is present in tobacco smoke and may be ubiquitous in the environment. As a biological monitor of carcinogen exposure, we have developed an immunological method for measuring the predominant carcinogen-DNA adduct of ABP, N-(deoxyguanosin-8-yl)-ABP (dG-C8-ABP). Rabbits were immunized with keyhole limpet hemocyanin (KLH) conjugate prepared by a periodate oxidation and coupling of N-(guanosin-8-yl)-ABP (rG-C8-ABP) to the protein. The resulting polyclonal antisera was systematically characterized using dual inhibitor methodology augmented by specialized computer and software support; and a competitive avidin-biotin enzyme-linked immunoassay (A-B ELISA) assay employing polyclonal rabbit anti-KLH-(rG-C8-ABP) was developed. Under the assay conditions described, the detection limit for dG-C8-ABP was 18 fmol/well. The relative lack of reactivity toward ABP, N-acetyl-4-aminobiphenyl, N-(deoxyadenosin-8-yl)-ABP, N-(deoxyguanosin-8-yl)-2-aminofluorene and deoxyguanosine as inhibitors indicated that primary specificity involves epitopes found on the purine and biphenyl rings. Results emphasize the need to define polyclonal anti-adduct sera operationally in the context of the antigen/assay system used to evaluate it. Assay sensitivity was achieved by decreasing the amount of antibody and solid-phase antigen in the competitive portion of the assay and the use of avidin-biotin as well as enzymatic amplification. This methodology is a useful alternative to other ultrasensitive techniques and should be directly applicable to the detection of ABP-DNA adducts in exposed human populations.

Isoniazid hepatotoxicity: Progress in understanding the immunologic component

The report by Metushi et al. “Detection of Anti-Isoniazid and Anti-CYP Antibodies in Patients with Isoniazid-Induced Liver Failure” addresses the role of the immune response in isoniazid (INH) mediated liver injury. Isoniazid is an essential drug in the treatment of tuberculosis. It is used as one component of combination therapy for the treatment of tuberculosis and as prophylaxis for patients deemed to have been exposed to tuberculosis.(1) While INH is generally considered to be safe and cost-effective, liver-related adverse drug reactions may occur and involve two forms of liver injury. Mild hepatitis is typically asymptomatic and self-limiting, occurs in the early stages of a course of treatment, and is thought to occur in approximately 10% of patients receiving monotherapy. Alanine aminotransferase (ALT) elevations are generally less than threefold the upper limit of normal. Overt hepatitis occurs in 0.5–1% of patients receiving INH as monotherapy, generally occurs early in the course of therapy, and is associated with gastrointestinal complaints, jaundice, and progression to liver failure.(1) Identified risk factors for INH acute liver failure (ALF) include female gender, age > 35 years, concomitant treatment with other anti-tuberculous drugs, slow acetylator NAT2 genotype (NAT2*7), cytochrome P450 2E1 (CYP 2E1 c1/c1) genotype, daily dose of INH > 50 mg/kg, concomitant treatment with acetaminophen, and daily ethanol use.(2) n nWhile the metabolism of INH (ie., oxidation of INH to a reactive metabolite) is known to contribute to the development of toxicity,(3, 4) the potential role of the immune system in the development of liver injury is unclear. Previous clinical and laboratory data have been inconclusive in this area, because INH liver injury is not universally associated with fever or rash, does not necessarily recur with rechallenge, and anti-INH antibodies have not been detected in previous studies.(2) However, new data,(5) including Metushi’s report in this issue of Hepatology, contribute to an evolving understanding regarding the contribution of the immune response to the etiology of INH hepatotoxicity.(5) n nUtilizing various immunoassay approaches, Metushi and colleagues measured antibodies to INH and native proteins in clinical samples from two patient groups. Patient samples (n=19) obtained from the Acute Liver Failure Study Group (ALFSG) registry deemed to have at least probable or higher (>50%) likelihood of toxicity due to INH represented the first group. Comparison samples were obtained from patients receiving INH prophylaxis (n=20), of whom 15 had no liver injury response, while five developed mild hepatitis (ALT 47-147 IU/L). Baseline demographic characteristics differed between the two groups by gender (73.7% female, ALFSG group vs. 40% female, prophylaxis group), while mean age was comparable between the groups. An antigen for detection of anti-INH antibody in patient’s serum was prepared by modifying lysozyme with the N-hydroxysuccinimide ester of isonicotinic acid (INA).(2) To test the specificity of the patient’s antibody to INH, free INH was mixed with patient serum. n nWestern blot assays showed inhibition of binding, confirming the assay’s specificity for INH. Samples from the two patient groups were subsequently tested with the synthetic Lys-INH antigen. Eight of the 19 samples from patients with INH ALF were positive (42%), while the samples from the INH prophylaxis group were all negative for antibodies to LYS-INH. n nAdditional assays were performed in the ALFSG samples to examine the immune response to native proteins involved in the metabolism of INH. These assays showed that a significant fraction (14 of 19) of ALFSG samples contained antibodies that reacted with one or more of the cytochromes P450, specifically CYP2E1, CYP3A4, or CYP2C9. These CYP P450 isoforms activate INH, as confirmed in this report and previously supported by clinical reports showing that INH inhibits the metabolism of known pharmacologic substrates of CYP2E1, CYP3A4, and CYP2C9.(6) While some of the patient samples contained antibodies to INH and several of the CYP P450’s, the pattern of detection of the various antigens was variable among the eight patients with positivity to INH. In addition, the ALFSG samples were tested with a composite antigen comprised of CYP-2E1 modified with INA to produce CYP-2E1-INH. Thus, the composite antigen contained antigenic determinants that would be recognized by the same antibodies that recognize LYS or LYS-INH and/or CYP-2E1 protein. Some patient samples (eg., ALF-3 and ALF-10) were negative for anti-CYP-2E1, but were positive for anti INH (LYS-INH) and CYP-2E1-INH. In this context, the positive reaction of select patients (ALF 1,6,13,15,16,17) to the CYP-2E1-INH composite antigen appears to be due to reactivity with the 2E1 protein antigenic determinants; the positive reaction of patient ALF 7 appears to be due to reactivity with the INH antigenic determinant; and the positive reaction of other patients (ALF-3,10,14,18, 19) could be due to either of both antigenic determinants. This concept could be ultimately confirmed in the future by competitive inhibition studies. For example, one could predict that the anti-CYP-2E1-INH reactivity of ALF 1,6,13,15,16,17 could be inhibited by incubating the antibody with free INH. n nThe overall conclusion of the Metushi study is consistent with the concept that in some INH hepatotoxicity patients, CYPs bioactivate INH to a reactive metabolite that covalently modifies the CYP and/or other native proteins, and that in some (as yet undefined) circumstances, this reaction constitutes an immunogenic experience, and that the resultant immune response may have specificity for the INH hapten, CYPs, or both. While the Metushi data adds support for an immune mechanism of injury, there is no proof that the detected anti-INH antibodies are associated with injury. The anti-INH antibodies and autoantibodies may be the result of a non-pathogenic immune response associated with INH-metabolism, but not associated with injury. Additional, larger studies are needed to more fully examine the relationship of detectable immune responses to LYS-INH and relevant CYP P450’s in relationship to the onset and degree of liver injury, with appropriate comparison to non-INH exposed human samples and INH prophylaxis samples. In addition, additional work is needed to examine whether or not the immune response to the CYP P450’s examined in this report (CYP2E1, CYP3A4, CYP2C9) with activity to INH is limited to these particular CYP P450 isoforms. For example, INH also interacts with CYP2C19, a highly polymorphic enzyme involved in the metabolism of a large number of drugs used clinically such as phenytoin.(6) The drug-drug interaction of INH and phenytoin is well documented and represents a clinically important issue for patients receiving INH, due to the potential for phenytoin toxicity secondary to the inhibitory effects of INH on the metabolism of phenytoin. In vitro studies have shown that INH potently inhibits the catalytic activity of CYP2C19 and CYP3A in a concentration dependent manner, while it is a non-competitive inhibitor of CYP2E1.(6) Other drug interactions with potential relevance in the clinical setting of INH hepatotoxicity include the concurrent use of INH with CYP2C19 substrates (eg., omeprazole, diazepam, citralopram, nelfenavir), CYP3A4 substrates (eg., carbamazepine, ethosuximide, vincristine) and/or CYP2E1 substrates (acetaminophen, ethanol).(6, 7) n nClinical studies of drug induced liver injury are very challenging due to the rare and the multi-factorial nature of the condition and the lack of mechanism-based analytical assays. Future strategies involving the collaborative use of international clinical sample banks - representing broad genetic diversity - would broaden and facilitate ongoing research in this area. An analysis of INH antibody positivity by ethnicity, drug metabolism genotype status (eg., N-acetylation, CYP P450), concomitant use of other medications, and the inclusion of appropriate negative controls would help to advance our growing understanding of the role of the immune system and its interaction with drug metabolism in the pathogenesis of INH hepatotoxicity. Ultimately, a strategy that incorporates new analytical approaches - addressing both the immune response and pharmacogenetic vulnerability - can be envisioned.