Metabolism of oxysterols derived from nonenzymatic oxidation of 7-dehydrocholesterol in cells.

Recent studies suggest that 7-dehydrocholesterol (7-DHC)-derived oxysterols play important roles in the pathophysiology of Smith-Lemli-Opitz syndrome (SLOS), a metabolic disorder that is caused by defective 3β-hydroxysterol-Δ(7)-reductase (DHCR7). Although 14 oxysterols have been identified as the primary products of 7-DHC autoxidation in organic solution, the metabolic fate of these oxysterols in a biological environment has not yet been elucidated. Therefore, we incubated these primary 7-DHC oxysterols in control Neuro2a and control human fibroblast cells and identified metabolites of these oxysterols by HPLC-MS. We also incubated Dhcr7-deficient Neuro2a cells and fibroblasts from SLOS patients with isotopically labeled 7-DHC (d(7)-7-DHC). The observation of matching d(0)- and d(7) peaks in HPLC-MS confirmed the presence of true metabolites of 7-DHC after excluding the possibility of ex vivo oxidation. The metabolites of primary 7-DHC oxysterols were found to contribute to the majority of the metabolic profile of 7-DHC in cells. Furthermore, based on this new data, we identified three new 7-DHC-derived metabolites in the brain of Dhcr7-KO mice. Our studies suggest that 7-DHC peroxidation is a major source of oxysterols observed in cells and in vivo and that the stable metabolites of primary 7-DHC oxysterols can be used as markers of 7-DHC peroxidation in these biological systems.

human fi broblasts (HF) were purchased from the Coriell Institute. Dhcr7 -defi cient Neuro2a cells were generated as described previously ( 22 ). All cell lines were maintained in DMEM supplemented with L-glutamine, 10% fetal bovine serum (FBS; Thermo Scientifi c HyClone, Logan, UT), and penicillin/streptomycin at 37°C and 5% CO 2 . Generally for different treatment, the HF cells were plated at a density of 3.0 × 10 5 cells/plate in 60 mm cell culture dishes (Sarstedt, Newton, NC), whereas the Neuro2a cells were plated at a density of ‫ف‬ 1 × 10 6 cells/plate in 100 mm dishes. Both cell lines were allowed 12 h to attach to the dishes. For treatment of Dhcr7 -defi cient Neuro2a cells or SLOS HFs, cells were cultured in the presence of different concentrations of d 7 -7-DHC in DMEM medium containing 10% lipid-reduced serum (HyClone Lipid Reduced FBS, Thermo Fisher Scientifi c Inc.), L-glutamine, and penicillin/streptomycin for fi ve days (medium was refreshed every two days). This lipid-reduced FBS does not contain a detectable level of cholesterol.
When treating control Neuro2a cells or control HF cells with different primary 7-DHC-derived oxysterols, the cells were cultured in the presence of different oxysterols (1 M for compounds 2a , 2b , and 3 , and 5 M for other oxysterols due to their different toxicity) ( 20 ) in medium with 10% regular FBS. At the end of the experiments, the cells were washed with 1× PBS buffer twice, scraped with a cell scraper, and centrifuged at 250 g for 5 min at 4°C. The cell pellets were frozen at Ϫ 80°C until lipid extraction and separation.

Dhcr7 -KO mice
Dhcr7 -KO ( Dhcr7 tm1Gst/J ) mice were purchased from Jackson Laboratories (catalog #007453). The mice were kept and bred in Division of Animal Care facilities at Vanderbilt University. All experimental procedures were in accordance with the National Institutes of Health guidelines for the use of live animals and were approved by the Vanderbilt University Institutional Animal Care and Use Committee. Genotyping and dissection of the tissues from P0 or E20 mice were performed as previously described ( 14 ). The brain tissues were rapidly removed and frozen in precooled methyl-butane and stored at Ϫ 80°C until lipid extraction and HPLC-APCI-MS/MS analysis.

Lipid extraction, separation, and HPLC-APCI-MS/MS analyses
Extraction and separation of the lipids from cell pellets and brain tissues were described in detail previously ( 14 ). The cells treated with d 7 -7-DHC were processed in the absence of any deuterated oxysterol standard, and the cells treated with 7-DHCderived oxysterols were processed in the presence of an appropriate amount of d 7 -DHCEO standard. Briefl y, to the cell pellets was added Folch solution [5 ml chloroform/methanol (2/1) containing 0.001M BHT and PPh 3 ], followed by the addition of NaCl aqueous solution (0.9%, 1 ml). The resulting mixture was vortexed for 1 min and centrifuged for 5 min. The lower organic phase was recovered, dried under a stream of nitrogen, redissolved in methylene chloride, and subjected to separation with NH 2 -SPE [500 mg cartridge; the column was conditioned with 4 ml of hexanes, and the neutral lipids containing oxysterols were eluted with 4 ml of chloroform/2-propanol (2/1)]. The eluted fractions were then dried under nitrogen and reconstituted in methylene chloride (200 l) for normal phase (NP) HPLC-APCI-MS/MS analyses following a previously reported method ( 13,20 ). HPLC was performed with either a 5 m 250 × 4.6 mm silica column (Beckman Coulter Inc.) or a 3 m 150 × 4.6 mm silica column (Phenomenex Inc.). A gradient of 2-propanol in hexanes was used as the elution solvent at 1.0 ml/min: 2-propanol/hexanes = 10/90 (0-15 min), 19/81 (16-26 min), 10/90 (27-35 min). MS analysis was performed on a Thermofi nnigan TSQ Quantum Ultra Oxysterols as a class can exert a broad spectrum of biological functions in cells and tissues. Among the functions affected are cell apoptosis, activation of the infl ammatory response, regulating cholesterol homeostasis and hedgehog signaling pathways, and modulating immune system responses (17)(18)(19). Individual oxysterols formed from free radical oxidation of 7-DHC exert differential cytotoxicity to cells, and the oxysterol mixture leads to changes in cell proliferation, differentiation, and gene expression ( 20 ). One of the major 7-DHC-derived oxysterols observed in cell and animal models of SLOS, 3 ␤ ,5 ␣ -dihydroxycholest-7-en-6-one (DHCEO), induces critical gene expression changes that relate to cell growth and lipid biosynthesis ( 14,20 ). This compound also accelerates differentiation and arborization of cortical neurons ( 21 ). Mechanistic studies in Neuro2a cells suggested that DHCEO is formed from one of the primary oxysterols of 7-DHC peroxidation, 7-DHC-5 ␣ ,6 ␣epoxide, via the intermediate 7-cholesten-3 ␤ ,5 ␣ ,6 ␤ -triol ( 14 ). However, the mechanisms of formation of other endogenous oxysterols, as well as the metabolic fate of other primary 7-DHC oxysterols, are still unknown.
We have investigated the metabolism of 7-DHC and its oxysterols in cell cultures and we report the following here: i ) the elucidation of the metabolic profi le of 7-DHC in Dhcr7 -defi cient Neuro2a cells and SLOS human fi broblasts by incubating deuterium-labeled 7-DHC ( d 7 -7-DHC) in these cell lines followed by HPLC-MS analysis; ii ) identification of the metabolites of primary oxysterols of 7-DHC autoxidation in control Neuro2a cells and control human fi broblasts by HPLC-MS; iii ) correlation of the metabolites of the primary 7-DHC oxysterols to the overall metabolic profi les of 7-DHC in Dhcr7 -defi cient Neuro2a cells and SLOS human fi broblasts; and iv ) identifi cation of three new 7-DHC-derived oxysterols in brain of Dhcr7 -KO mice based on the metabolic profi le of primary 7-DHC oxysterols.

Cell cultures and treatment of human fi broblasts and Neuro2a cells
Neuroblastoma cell line Neuro2a was purchased from the American Type Culture Collection (Rockville, MD). Control (GM05399 and GM05565) and SLOS (GM05788 and GM03044) with synthetic standards of these compounds ( 13,15 ). The structures of these oxysterols are shown in Fig. 2 . Furthermore, two unknown 7-DHC-derived peaks were observed in the m/z panels that correspond to [7-DHC+1O+H + ] and [7-DHC+1O+H + -H 2 O] (unknown-1 and unknown-2). Because 7-DHC is exceptionally oxidizable, we sought to exclude the possibility that the observed oxysterols were formed during sample collection and preparation. We processed control samples of Dhcr7 -defi cient Neuro2a cells (grown in the absence of d 7 -7-DHC) in the presence of externally added d 7 -7-DHC and compared the ratios of the d 7 -and corresponding d 0 -oxysterols to the ratio of d 7 -and d 0 -7-DHC in the cells ( Fig. 3 ). If the ratio of d 7 / d 0 determined for a given oxysterol was signifi cantly less than the measured d 7 -/ d 0 -7-DHC ratio, the oxysterol was likely formed in the live cell. If the d 7 / d 0 ratio of an oxysterol is comparable to the d 7 / d 0 ratio of its 7-DHC precursor, then this oxysterol was formed during sample processing. From this experiment, we confi rmed that all 4 ␣ -hydroxy-7-DHC, 4 ␤ -hydroxy-7-DHC, and unknown-2; a majority of DHCEO, THCEO, DHCDO, and 7-kDHC; and ‫ف‬ 50% of unknown-1 are formed in cells, suggesting that these oxysterols are indeed metabolites of 7-DHC in cells.

Oxysterol metabolic profi le of 7-DHC in SLOS human fi broblasts
The metabolic profi le of 7-DHC in SLOS human fi broblasts is similar to the one observed for Dhcr7 -defi cient Neu-ro2a cells, but with DHCEO being the major metabolite present ( Fig. 1B ). Specifi cally, 4 ␣ -hydroxy-and 4 ␤ -hydroxy-7-DHC were only observed as very minor metabolites in fibroblasts and the unknown peak (unknown-2) in the m/z panel of [7-DHC+1O+H + ] was not observed.
Although the metabolic profi le is similar between Dhcr7defi cient Neuro2a cells and SLOS human fi broblasts, there are clear differences in the way metabolites are formed, as indicated by the d 7 / d 0 ratio of each metabolite. In human fi broblasts, every metabolite has a larger d 7 / d 0 ratio than the d 7 / d 0 ratio of 7-DHC ( Fig. 4A ). This is not surprising since d 7 -7-DHC is present at the beginning of the cell treatment (allowing d 7 -oxysterol formation at an earlier stage), but d 0 -7-DHC accumulates slowly by biosynthesis over the course of incubation in lipid-reduced medium. However, in Dhcr7 -defi cient Neuro2a cells, only the d 7 / d 0 ratios of DHCEO, 7-kDHC, THCEO, DHCDO, and unknown-1 were larger than that of 7-DHC. The d 7 / d 0 ratios of 4 ␣ -hydroxy-7-DHC, 4 ␤ -hydroxy-7-DHC and unknown-2 were not different from the same ratio of 7-DHC ( Fig. 4B ).
Another metabolic difference between these two cell lines is that d 7 -7-DHC was converted to d 7 -cholesterol to different extents. The d 7 / d 0 ratio of cholesterol is comparable to that of 7-DHC in Dhcr7 -defi cient Neuro2a cells (0.033 versus 0.038), but is only ca. 6% of the same ratio of 7-DHC in SLOS human fi broblasts (0.011 versus 0.18). This difference is presumably due to the fact that the Dhcr7defi cient (knockdown by shRNA) Neuro2a cells still retain some activities of Dhcr7 ( 22 ). ) in the mass spectrometry ( 14,15 ).

Oxysterol metabolic profi le of 7-DHC in Dhcr7-defi cient Neuro2a cells
We reported previously that a number of HPLC-MS fractions that have molecular mass corresponding to oxysterols have been found in the analysis of lipid from Dhcr7 -deficient Neuro2a cells and SLOS human fi broblasts, when the analysis was compared with matching control samples. However, only one of these new peaks, DHCEO, has been identifi ed ( 14 ). To identify the additional unknown peaks, we incubated these two cell lines in medium containing lipid-reduced serum supplemented with d 7 -7-DHC, and we analyzed the oxysterols in the cells by HPLC-MS (see Materials and Methods).

Oxysterol metabolic profi le of primary nonenzymatically formed 7-DHC-derived oxysterols in control Neuro2a cells and control human fi broblasts
We presume that primary nonenzymatically formed 7-DHC oxysterols are further metabolized to more stable oxysterols in cells and tissues. To examine this hypothesis, we incubated major primary 7-DHC autoxidation-derived oxysterols and cholesta-7-en-3 ␤ ,5 ␣ ,6 ␣ ( ␤ ),9 ␣ -tetraols [6 ␣ ( ␤ )tetraols] ( Fig. 5 ) in control Neuro2a cells and control human fi broblasts, and we analyzed the metabolites of these oxysterols by HPLC-MS. Noncytotoxic concentrations of oxysterols (1 M for compounds 2a and 2b , and 5 M for other compounds) were used in this study based on our previous study in Neuro2a cells ( 20 ). At these concentrations, the oxysterols did not induce changes in the cell viability and morphology of these two cell lines. The same amount of d 7 -DHCEO was added to each sample during  alcohol, but the 6 ␤ -tetraol is clearly more prone to allylic oxidation as indicated by the formation of much higher level of THCEO from this substrate than from the 6 ␣ compound under the same conditions ( Fig. 6E, F ). In addition, the allylic alcohol at C6 having a double bond at C7 = C8 appears to be much more reactive toward oxidation than the one at C7 with the double bond at C8 = C14 (from metabolism of compounds 6a and 6b ) as no ketone products were observed as metabolites of compounds 6a and 6b (no peak with a m/z value of the potential ketone product was observed) (supplementary Fig. II).

Identifi cation of THCEO, DHCDO, and 7-kDHC in brain of Dhcr7-KO mice
To establish whether the oxysterol profi le in the nervous system refl ects the profi le identifi ed in the neuronal cell line, we examined the oxysterols formed in brain of Dhcr7 -KO mice at P0 (E20). Although we have previously identifi ed several oxysterols in brain of the KO mice, [including 24-hydroxy-7-DHC, DHCEO, 4 ␣ -hydroxy-7-DHC, and 4 ␤ -hydroxy-7-DHC ( 16 )], several oxysterols remained unidentifi ed. With the knowledge obtained from our studies of oxysterol metabolism in cells, we found three previously unidentifi ed oxysterols, THCEO, DHCDO, and 7-kDHC, based on the retention time and MS characteristics by comparing with the standards of these oxysterols and d 7 -DHCEO ( Fig. 8B ). A few minor peaks remain unidentifi ed due to lack of matching standards. No 7-DHC-derived oxysterol was observed in brain of wild-type mice ( Fig. 8A ). At E20 or P0, the identifi ed oxysterols refl ect primarily neuronal metabolism because glial cell development and myelination proceed postnatally ( 24,25 ). Therefore, the presence of these identifi ed oxysterols suggests that 7-DHC peroxidation in neurons proceeds in a way similar to that in the Neuro2a cell line. Future studies in hypomorphic mouse models will allow us to compare glial and neuronal metabolism of 7-DHC-derived oxysterols ( 26 ).

DISCUSSION
Free radical oxidation of 7-DHC in solution gives over a dozen oxysterols as products, and these oxysterols are found to exert a variety of biological activities, including cytotoxicity and induction of gene expression changes ( 13,20,21 ). However, the oxysterol profi les found in cell and rodent models of SLOS are distinctly different from the peroxidation product profi le observed from authentic free radical oxidation in organic solvent. Our studies here suggest that the primary oxysterols formed from 7-DHC peroxidation are further metabolized in biological environments, leading to the observed differences in the oxysterol profi les.
The studies reported here clearly establish links between the primary oxysterols derived from 7-DHC autoxidation and the oxysterol metabolites observed in cell and animal SLOS models. The incubation experiments in Dhcr7defi cient Neuro2a cells and SLOS human fi broblasts with d 7 -7-DHC ( Fig. 1 ) and the ex vivo oxidation tests ( Fig. 3 ) ring opening, and/or dehydration (supplementary There are some differences in the reactivities of different oxysterols for the similar metabolic transformations. For example, the 6 ␣ -and 6 ␤ -tetraols both contain an allylic Fig. 3. Evaluation of oxysterol formation during sample processing of Dhcr7 -defi cient Neuro2a. Cell samples were cultured in the absence of d 7 -7-DHC but processed in the presence of externally added d 7 -7-DHC. The ratio of d 7 -/ d 0 -oxysterol is compared with the same ratio of 7-DHC. All ratios of d 7 -/ d 0 -oxysterols were found to be signifi cantly less than the ratio of d 7 -/ d 0 -7-DHC. Statistical analyses were performed with t -test (two-tailed distribution) against d 7 / d 0 ratio of 7-DHC. N = 3; * P < 0.05; *** P < 0.0005. of rodent models of SLOS ( 15,16 ), and they are likely formed from enzymatic oxidation of 7-DHC as they were not isolated as products of free radical oxidation of 7-DHC in solution ( 13 ). However, the origin of these two oxysterols is still debatable as 4 ␤ -hydroxycholesterol, but not unequivocally identifi ed the metabolites that are derived from 7-DHC in cell culture. The identifi ed products include 4 ␣ -hydroxy-7-DHC, 4 ␤ -hydroxy-7-DHC, 7-kDHC, DHCEO, THCEO, and DHCDO ( Fig. 2 ). 4 ␣ -Hydroxy-7-DHC and 4 ␤ -hydroxy-7-DHC have been identifi ed in tissues  The primary oxysterols formed from 7-DHC autoxidation in solution are biologically active in control Neuro2a cells and in cortical neuronal cells, being cytotoxic and inducing changes in gene expression and morphology ( 20,21 ). In Neuro2a cells, a mixture of the primary 7-DHC oxysterols downregulates gene transcripts related to lipid biosynthesis (including Hmgcr , Dhcr7 , Srebp2 , Fasn , S1p , and Sqs ) and cell growth (including Ki67 , Adam19 , and Egr1 ) ( 20 ). While the stable metabolites of the primary oxysterols can be used as biomarkers of 7-DHC peroxidation, they may by themselves exert various biological activities. For example, DHCEO is not only toxic to neuronal cells but also induces changes in critical gene transcripts ( 20 ). Accelerated 4 ␣ -hydroxycholesterol, was identifi ed as an enzymatic oxidation product of cholesterol via the catalysis of CYP 3A4 ( 27,28 ). More importantly, DHCEO and THCEO are also identifi ed as the stable end metabolic products of primary oxysterols of 7-DHC peroxidation, suggesting that peroxidation of 7-DHC contributes signifi cantly to the overall oxysterol pool in cell culture and in vivo ( Figs. 1 and 6 ). We note that DHCEO and THCEO are very minor products of 7-DHC autoxidation, yet they are among the major metabolites observed in cells and in vivo. This is perhaps not surprising since their precursors, 7-DHC-5 ␣ ,6 ␣epoxide and compounds 1 , 2a , and 2b , are major primary oxysterols formed from 7-DHC autoxidation ( 13 ).   ( 15,16 ), while THCEO, DHCDO, and 7-kDHC were identifi ed in this study. A known amount of d 7 -DHCEO was added to each sample as an internal standard so that retention time and peak intensities of the peaks could be compared between each run. New and unidentifi ed peaks observed in KO mice relative to WT are marked with an asterisk. 5 ␣ ,8 ␣ -Epidioxycholest-6-en-3 ␤ -ol (EnP) ( 5,8 ) is a photooxidation product of 7-DHC that has been demonstrated to be a product of ex vivo oxidation ( 15 ). The parent ion of each chromatogram and the MS/MS transition are marked in the corresponding MS panel. cell differentiation has also been observed in both Neuro2a cells and primary neurons treated with DHCEO ( 20,21 ).
The observed d 7 / d 0 ratios of the oxysterols shed light on the differences in metabolism between the two different cell lines studied. As seen in Fig. 4 , the d 7 / d 0 ratios of all oxysterols in human fi broblasts are larger than the d 7 / d 0 ratio of the precursor 7-DHC, which is reasonable considering d 7 -7-DHC is present at high concentrations even during the early stages of treatment. In addition, the d 7 / d 0 ratios are quite similar among all oxysterols, suggesting that d 7 -7-DHC is essentially equivalent to d 0 -7-DHC during its metabolism in human fi broblasts. In Neuro2a cells, most of the d 7 / d 0 -oxysterol ratios are larger than the 7-DHC ratios, but 4 ␣ -hydroxy-7-DHC, 4 ␤ -hydroxy-7-DHC, and unknown-2 have ratios comparable to that of 7-DHC ( Fig. 4B ), suggesting that exogenously added d 7 -7-DHC and metabolically derived d 0 -7-DHC are differentiated by some of the subsequent metabolic pathways. Thus, it is reasonable to speculate that the differences in oxysterol profi les between these two cell lines may be due to i ) their different metabolic activities, as Neuro2a is an immortal neuroblastoma cell line and human fi broblast is a primary cell line; ii ) their different expression of enzymes responsible for the metabolic transformations; iii ) their different antioxidant capacity; and iv ) different access of endogenous d 0 -and exogenous d 7 -7-DHC to the necessary enzymes. For example, it is known that cholesterol is mainly synthesized in endoplasmic reticulum in cells ( 35 ), whereas the exogenously added d 7 -7-DHC presumably resides mostly in the membrane.
The metabolites of the primary nonenzymatically formed 7-DHC oxysterols are also observed in the brain of Dhcr7 -KO mice. In addition to the known DHCEO ( 14-16 ), we identifi ed THCEO, DHCDO, and 7-kDHC in this study. Note that the oxysterol profi les between Dhcr7 -defi cent Neuro2a cells and the Dhcr7 -KO brain are quite similar, suggesting that Neuro2a cells are a good model to study