The main aim of this study was to demonstrate the feasibility of using metabolomics on intracellular compounds of in vitro cell systems such as HepG2 cells, as an alternative approach to studying the effects of toxicants. We have demonstrated that reproducible results can be obtained and that differences between controls from different experiments can be subjected to statistical analysis, but that caution is required with regard to repeatability of experiments; generally, the same effects are observed in different experiments but their magnitudes can differ. However, there is a consistent effect (due to TCDD in this study) on metabolic profiles using an untargeted approach. The changes in metabolite concentrations are minimal and can vary because of the effect of the treatment on cell numbers. Therefore, normalization (using PL and PUFA), as described above, is crucial; so is having enough biological replicates to ensure the accuracy of the data. Without normalization or sufficient biological replicates, several of the metabolic changes may not have been detected. In apparent contradiction to the normalization using PL, it has been reported that TCDD induces the expression of the phospholipase A2α (PLA2α) gene in mouse hepatoma Hepa-1c1c7 . However, in this HepG2 study, degradation of PL was not evident with regard to total PL. Furthermore, a parallel study using transcriptomics did not provide evidence of an effect of TCDD on PLA2α gene expression in HepG2 cells; this supports the present findings for HepG2 cells and supports the validity of the chosen normalization method.
To substantiate the usefulness of the untargeted metabolomics methodology, the observed changes in metabolite levels were interpreted in the light of published data concerning the effect of TCDD. Ultimately, this was the validation of the procedures and protocols developed in this study. In general, a decrease or increase of an intracellular metabolite concentration is not necessarily an indicator of flux. Actual metabolite levels depend on how biochemical pathways/processes are regulated, how metabolites in pathways interact and how fast and in which directions reactions occur (kinetics). Cells tend to balance their metabolism after perturbation in order to maintain homeostasis. Therefore, it is likely that only changes in the main pools of substrates in the cell are measured when it is exposed to a toxicant. Inhibition/activation of a pathway may lead to increased or decreased levels of relevant pools of compounds. However, it is often difficult to determine whether inhibition or activation is relevant by observing metabolite concentrations at one time point. As discussed in more detail below, TCDD is known to have an effect on the proliferative capacity, metabolism and antioxidant status of the cell.
Effect on cell proliferation
Membrane constituents such as PL (Figure 2B) and free cholesterol are present at lower levels in extracts obtained after treatment with TCDD because there are fewer cells in the culture, and are indicative of decreased proliferation during exposure; the anti-proliferative effect of TCDD has been described previously . The lower levels of nucleotides in treated extracts indicate a change in metabolites known to be involved in DNA synthesis (Table 4).
Spermidine and N1-acetylspermidine were also found in lower levels after TCDD treatment. Polyamines have important functions in cell proliferation, differentiation and apoptosis, and they are essential for hepatic growth and regeneration . Polyamines can be synthesized in liver from agmatine (formed by decarboxylation of arginine, via arginine decarboxylase) , but ornithine decarboxylase (ODC) is the main enzyme responsible for polyamine biosynthesis. ODC is involved in the conversion of ornithine into putrescine, the precursor of other polyamines [38, 39]. Their catabolism, on the other hand, is regulated by the enzymatic action of the polyamines spermidine/spermine N1-acetyltransferase (cSAT) and diamine oxidase .
ODC is induced during conditions where there is enhanced gene activation and tissue growth. It has been reported that ODC induction is inhibited by TCDD [38, 39].
Therefore, it is possible that induction of polyamine synthesis in cultured HepG2 cells was inhibited by TCDD, resulting in the low content of spermidine.
Effect on metabolism
Changes in metabolic profiles due to TCDD treatment of HepG2 cells indicates major changes in the general metabolism of these cells, which involves fatty acids, amino acids and nucleotides. This is reminiscent of wasting syndrome [21, 41–43].
The decreases in triglycerides, cholesterol ester and unsaturated fatty acids observed in the 1H NMR analysis of the apolar fraction, together with the decreases in several fatty acids observed in the GC-MS data, indicate that under the conditions used in this study, TCDD affected lipid metabolism.
Several in vivo studies [44–46] have demonstrated that TCDD reduces the activity of enzymes involved in the synthesis of de novo fatty acids (such as fatty acid synthase, FAS and acetyl CoA carboxylase, AAC) and in cholesterol biosynthesis (such as 3-hydroxy-methylglutaryl-CoA synthase) or the expression of genes encoding these enzymes [41, 42, 47–49]. This effect, which was also observed in experiments using mouse embryo fibroblasts , supports the results presented here. In studies carried out in vivo  and in vitro , it has been reported that the effect of TCDD on the gene expression of enzymes involved in the de novo synthesis of fatty acids is mediated by AhR.
Using GC-MS analysis it is possible to determine the nature of the fatty acids that are most affected by exposure to TCDD (Table 2). The levels of the majority of fatty acids decreased upon treatment. However, the contents of heptadecanoic (C17:0) and octadecanoic, i.e. stearic (C18:0), acids were increased after exposure. The increased percentage of saturated and the decreased percentage of mono-unsaturated fatty acids could be due to the decreased expression of stearoyl-CoA-desaturase (SCD1), as demonstrated in mouse embryo fibroblasts after TCDD exposure . SCD1 is the rate limiting enzyme in the biosynthesis of mono-unsaturated fatty acids and catalyzes the introduction of the cis double bond in the Δ9 position of acyl CoA substrates . Therefore, inhibiting the expression of SCD1 using TCDD would partially explain the increased ratio between saturated/monounsaturated fatty acids observed in this study.
Pantothenic acid and AMP are metabolites found in lower concentrations after exposure to TCDD (Table 4). Pantothenic acid and AMP are two of the three precursors for coenzyme A (CoA) synthesis. In general, during fatty acid degradation and synthesis, CoA transports fatty acids as acyl groups through repetitive degradative or synthetic cycles.
Furthermore, decreases in the content of propionyl- and butyryl-carnitines were observed when cells were exposed to TCDD. These short chain acyl-carnitines are metabolic products of the reaction of acyl CoA and carnitine, which is mediated by transferases in the mitochondria . Formation of butyryl- and propionyl-carnitines is favored when butyryl and propionyl-CoA accumulate in the mitochondria owing to an increased acyl-CoA/CoA ratio. In the case of propionyl-CoA, an increase in the catabolism of amino acids such as valine, isoleucine and - indirectly - threonine and methionine also favors its formation . Changes in these carnitine derivatives indicate that mitochondrial activity and transport across mitochondrial membranes is affected by CoA modulation and/or beta-oxidation of fatty acids.
The level of citrate increased, whereas the level of lactate decreased, after exposure. Citrate is a feedback inhibitor for glycolysis at the level of phosphofructokinase. Lactate is a product of the conversion of pyruvate by lactate dehydrogenase. These two metabolite changes can indicate that glycolytic activity is lower because of an influx of acetyl-CoA, for instance due to fatty acid beta-oxidation. This is in agreement with the breakdown of triglycerides and degradation of fatty acids, and changed activity in mitochondria (see above).
Together, these metabolic effects are reminiscent of a wasting syndrome in which fatty acid metabolism and energy metabolism (glycolysis, TCA cycle) are affected, and the amino acid and nucleic acid pools are reduced. These latter effects could contribute to the observed decrease in cell proliferation. However, it is not clear from the data whether beta-oxidation increased or if fatty acids were transported out of the cell. In adipose tissue, TCDD mobilizes fatty acids towards the plasma [41, 42, 46, 55, 56]. An increase in amino acids circulating in plasma owing to TCDD exposure has previously been reported . Therefore, the data could imply a shut-down of the cells followed by the export of several metabolites.
Several in vivo studies demonstrate an accumulation of lipids in the liver [21, 46, 55, 58], although the effects of TCDD vary depending on the dose, exposure-time or test animal used [41, 44, 45, 59]. An increase in liver lipid content is in contrast to the results found in this research. However, in the published studies, a loss in body fat is demonstrated, as is an increase in the serum lipid content [41, 42, 46, 55, 56]. The increased serum lipid content could increase uptake and accumulation of extrahepatic lipids in liver [46, 60]. This would explain the occurrence of a fatty liver in vivo, and the differences in terms of lipid content between in vivo and in vitro studies.
Effect on antioxidant status
NMR and LC-MS spectra demonstrated an increase in reduced (GSH) and oxidized (GSSG) glutathione content in samples treated with TCDD (Table 4). However, it has been reported that GSH content decreases after exposure to TCDD . Furthermore, from the 1H NMR data, it was observed that the GSH/GSSG ratio increased ca. 20% with TCDD exposure. This ratio, considered a parameter for measuring the oxidative status of a cell, has been reported to decrease in several in vivo studies as a consequence of exposure to TCDD [23, 61]. However, data are variable and some authors have reported an increase in the GSH/GSSG ratio after mice were treated with TCDD .
GSH acts as a nucleophilic "scavenger" of numerous compounds and their metabolites via enzymatic and chemical mechanisms (converting electrophilic centers to thioether bonds) and as a substrate in the glutathione peroxidase (GPx)-mediated reduction of lipid hydroperoxides and of H2O2 to water [62, 63]. As a consequence of this reaction, GSH is oxidized to GSSG, which is in turn recycled back to GSH via glutathione reductase (GR). Therefore, any effect on the activity of GPx, GR or both could affect the ratio between GSH and GSSG. Several in vivo  and in vitro [64, 65] studies have described the inhibition of GPx activity in liver due to TCDD treatment. Inhibition of the activity of this enzyme could theoretically lead to an increase in the content of GSH and in the GSH/GSSG ratio, as observed herein. However, it has been reported that the activity of GPx in liver mitochondria of mice exposed to TCDD is increased , but increased activity of GR in liver mitochondria was also evident, possibly explaining the increased GSH/GSSG ratio following dioxin treatment. Some studies have demonstrated that TCDD increases the activity of GR in liver cells , whereas other authors describe the opposite effect .
In relation to the synthesis of GSH, Boverhof and co-authors  demonstrated up-regulated expression of the genes for enzymes involved in the synthesis of GSH (such as glutamate-cysteine ligase, Gclc, and glutathione synthase, Gss) after rats and mice were exposed to TCDD. Increased synthesis of GSH could explain the results. Boverhof and co-authors  described a down-regulation of the genes associated with the metabolism of glutamate and glycine, which are building blocks of GSH. They suggested that this down-regulation could be an adaptation of the cell to conserve these amino acids for increased glutathione synthesis.
Other effects associated with TCDD treatment
Creatine is decreased in cells as a result of TCDD treatment (Table 4). In relation to this, Boverhof and co-authors  found that genes expressing guanidinoacetate N-methyltransferase (GAMT), one of the key enzymes in the biosynthesis of creatine, are down-regulated in liver after TCDD administration to rats. It has been reported that GAMT deficiency is associated with a reduction in body weight owing to reduced body fat mass , which could be related to the aforementioned in vivo effects of TCDD exposure.
The content of taurine, a non-protein sulfur-containing β-amino acid, is increased after the exposure of cells to TCDD. Taurine plays an important role in several biological processes including anti-oxidation, detoxification, membrane stabilization and maintenance of osmolarity . Moreover, it has been demonstrated that there is a relationship between taurine and lipid metabolism [69, 70]. Taurine lowers hepatic triglyceride concentration, reduces the synthesis of cellular cholesterol ester and elevates hepatic free fatty acids. These effects are in agreement with the results of the present study for HepG2 cells exposed to TCDD. TCDD down-regulates cysteine dioxygenase (CDO) [41, 42, 71, 72], the first enzyme in the conversion of cysteine to taurine. This seems to conflict with the observation of higher taurine levels after exposure to TCDD, but since the regulation of taurine concentration is unknown, perhaps the increased concentration of taurine after exposing cells to TCDD is related to other mechanisms.
An increased signal (average ratio TCDD: DMSO = 1.5) tentatively assigned to uridine diphosphate-N-acetylgalactosamine (UDP-NAcGal) and/or uridine diphosphate-N-acetylglucosamine (UDP-NacGlu) was observed after TCDD exposure (Table 4).
UDP-NAcGal and UDP-NacGlu are N-acetylhexosamines involved in several biosynthetic reactions including O-glycosylation of proteins on serine and threonine residues. O-linked glycosylation involves the transfer of the N-acetylhexosamine group from the UDP-acetylhexosamine, which is catalyzed by glycosyltransferases [73, 74].
Lower activity of these enzymes could theoretically produce an increase in the levels of UDP-N-acetylhexosamines. In agreement with this, decreased expression of GALNT1, the gene encoding the UDP-N-acetylgalactosamine transferase, was observed by Kim and co-authors  after HepG2 cells were exposed to TCDD.
Changes in the intracellular levels of UDP-N-acetylhexosamines, as observed in this study after exposure to TCDD, may indicate that protein O-glycosylation is affected. Altered expression levels of glycosylated protein have been described after TCDD exposure in vitro .
We have no explanation for the decrease in concentration of N-acetyl aspartate. This metabolite is normally expressed at high concentrations in the brain and has been implicated as an osmolyte  with the potential to bind to calcium ions or metal ions .