Letha H. Couch

Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry.

Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N-(nervonyl)-sphingosine were observed in an extract from 40,000 HL-60 cells. Perbenzoylation with benzoyl chloride permitted RPHPLC separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.

Photodecomposition of Pigment Yellow 74, a Pigment Used in Tattoo Inks¶

Tattooing has become a popular recreational practice among younger adults over the past decade. Although some of the pigments used in tattooing have been described, very little is known concerning the toxicology, phototoxicology or photochemistry of these pigments. Seven yellow tattoo inks were obtained from commercial sources and their pigments extracted, identified and quantitatively analyzed. The monoazo compound Pigment Yellow 74 (PY74; CI 11741) was found to be the major pigment in several of the tattoo inks. Solutions of commercial PY74 in tetrahydrofuran (THF) were deoxygenated using argon gas, and the photochemical reaction products were determined after exposure to simulated solar light generated by a filtered 6.5 kW xenon arc lamp. Spectrophotometric and high‐pressure liquid chromatography (HPLC) analyses indicated that PY74 photodecomposed to multiple products that were isolated using a combination of silica chromatography and reversed‐phase HPLC. Three of the major photodecomposition products were identified by nuclear magnetic resonance and mass spectrometry as N(2‐methoxyphenyl)‐3‐oxobutanamide (o‐acetoacetanisidide), 2‐(hydroxyimine)‐N‐(2‐methoxyphenyl)‐3‐oxobutanamide and N,N″‐bis(2‐methoxyphenyl)urea. These results demonstrate that PY74 is not photostable in THF and that photochemical lysis occurs at several sites in PY74 including the hydrazone and amide groups. The data also suggest that the use of PY74 in tattoo inks could potentially result in the formation of photolysis products, resulting in toxicity at the tattoo site after irradiation with sunlight or more intense light sources.

METABOLISM OF PIGMENT YELLOW 74 BY RAT AND HUMAN MICROSOMAL PROTEINS

Pigment Yellow 74 (PY74) is a monoazo pigment that is used in yellow tattoo inks. The metabolism of PY74 was investigated using rat liver and human liver microsomes and expressed human cytochromes P450 (P450s). Two phase I metabolites were isolated and characterized by mass spectrometry and NMR techniques. One metabolite (PY74-M1) was a ring hydroxylation product of PY74, 2-((2-methoxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxobutanamide. The second metabolite (PY74-M2) was identified as 2-((2-hydroxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxobutanamide, which is the O-demethylation product of PY74-M1. These metabolites were formed by in vitro incubations of PY74 with 3-methylcholanthrene-induced rat liver microsomes and to a much lesser extent by liver microsomes from untreated or phenobarbital-induced rats. The role for CYP1A in the metabolism of PY74 was confirmed using expressed human P450s. The catalytic ability of the P450s for metabolism of PY74 was CYP 1A2 > CYP 1A1 > CYP 3A4 ≈ CYP 1B1 (no activity with CYP 2B6, 2C9, 2D6 or 2E1). The metabolism of PY74-M1 to PY74-M2 was catalyzed only by CYP 1A2 and CYP 1A1 (no activity from CYP 1B1, 2B6, 2C9, 2D6, 2E1, or 3A4). These results demonstrate that the tattoo pigment PY74 is metabolized in vitro by P450 to metabolites that should be available for phase II metabolism and excretion.

Mitochondrial dysfunction induced by leflunomide and its active metabolite

Leflunomide, an anti-inflammatory drug used for the treatment of rheumatoid arthritis, has been marked with a black box warning regarding an increased risk of liver injury. The active metabolite of leflunomide, A771726, which also carries a boxed warning about potential hepatotoxicity, has been marketed as teriflunomide for the treatment of relapsing multiple sclerosis. Thus far, however, the mechanism of liver injury associated with the two drugs has remained elusive. In this study, cytotoxicity assays showed that ATP depletion and subsequent LDH release were induced in a time- and concentration-dependent manner by leflunomide in HepG2 cells, and to a lesser extent, by A77 1726. The decline of cellular ATP levels caused by leflunomide was dramatically exacerbated when galactose was substituted for glucose as the sugar source, indicating a potential mitochondrial liability of leflunomide. By measuring the activities of immuno-captured mitochondrial oxidative phosphorylation (OXPHOS) complexes, we found that leflunomide and A77 1726 preferentially targeted complex V (F1FO ATP synthase), with IC50 values of 35.0 and 63.7 μM, respectively. Bongkrekic acid, a mitochondrial permeability transition pore blocker that targets adenine nucleotide translocase, profoundly attenuated mitochondrial membrane depolarization, ATP depletion, and LDH leakage induced by leflunomide and A77 1726. Substantial alterations of mitochondrial function at the transcript level were observed in leflunomide-treated HepG2 cells, whereas the effects of A77 1726 on the cellular transcriptome were much less profound. Our results suggest that mitochondrial dysfunction may be implicated in the hepatotoxicity associated with leflunomide and A77 1726, with the former exhibiting higher toxicity potency.