Joyce A. Pepe

Effects of new anticonvulsant medications on porphyrin synthesis in cultured liver cells: Potential implications for patients with acute porphyria

Some patients with acute hereditary porphyrias have seizures and require anticonvulsant therapy, but many anticonvulsants induce exacerbations of the hepatic porphyrias. Recently, several new anticonvulsants have become available. Among these are gabapentin, vigabatrin, felbamate, lamotrigine, and tiagabine. Little is known about their potential for induction of porphyric attacks. We used a cell culture model of primary chicken embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of these new anticonvulsants on porphyrin accumulation. Treatment of the cells with deferoxamine (250 µM) led to a partial block in heme synthesis, simulating the conditions encountered in human beings with porphyria. Concomitant exposure of these cells to phenobarbital (2 mM) strongly induced accumulation of porphyrins, serving as a positive control in this model. Cells were treated for 20 hours with increasing doses (3.2 to 1,000 µM) of the newer anticonvulsants, with or without deferoxamine. For most of these anticonvulsants 5 to 100 µM is representative of the concentrations achieved in humans with therapeutic doses. Porphyrins were measured spectrofluorometrically as uro-, copro-, and protoporphyrins. Results were confirmed by high-pressure liquid chromatography. Neither vigabatrin nor gabapentin treatment, with or without deferoxamine, led to any increase in porphyrin accumulation. Similar doses of felbamate (with deferoxamine) led to a marked increase in (mainly proto-) porphyrin levels, qualitatively and quantitatively almost identical to the accumulation produced by phenobarbital. Lamotrigine or tiagabine (with deferoxamine) caused similar porphyrin accumulation. Tiagabine treatment up to 100 µM(with deferoxamine) also resulted in very high levels of predominantly proto-porphyrin. In contrast to the other anticonvulsants tested, tiagabine without deferoxamine led to mild porphyrin accumulation. In the presence of deferoxamine, phenobarbital, felbamate, lamotrigine, or tiagabine, but not gabapentin or vigabatrin, increased levels of the mRNA of ALA synthase, the first and rate-controlling enzyme of porphyrin synthesis. Such enzyme induction is a sine qua non for acute porphyric attacks. We conclude that neither vigabatrin nor gabapentin is porphyrogenic, whereas felbamate, lamotrigine, and, especially, tiagabine lead to much accumulation of porphyrins. The latter three anticonvulsants, therefore, may precipitate or exacerbate acute porphyric attacks in humans. We recommend use of vigabatrin or gabapentin, but not felbamate, lamotrigine, or tiagabine, in patients with acute porphyria and seizures.

Differential effects of metalloporphyrins on messenger RNA levels of delta-aminolevulinate synthase and heme oxygenase. Studies in cultured chick embryo liver cells.

The acute porphyrias in relapse are commonly treated with intravenous heme infusion to decrease the activity of delta-aminolevulinic acid synthase, normally the rate-controlling enzyme in heme biosynthesis. The biochemical effects of heme treatment are short-lived, probably due in part to heme-mediated induction of heme oxygenase, the rate-controlling enzyme for heme degradation. In this work, selected nonheme metalloporphyrins were screened for their ability to reduce delta-aminolevulinic acid synthase mRNA and induce heme oxygenase mRNA in chick embryo liver cell cultures. Of the metalloporphyrins tested, only zinc-mesoporphyrin reduced delta-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA. The combination of zinc-mesoporphyrin and heme, at nanomolar concentrations, decreased delta-aminolevulinic acid synthase mRNA in a dose-dependent manner. The combination of zinc-mesoporphyrin (50 nM) and heme (200 nM) decreased the half-life of the mRNA for delta-aminolevulinic acid synthase from 5.2 to 2.5 h, while a similar decrease was produced by heme (10 microM) alone (2.2 h). The ability of zinc-mesoporphyrin to supplement the reduction of delta-aminolevulinic acid synthase mRNA by heme, in a process similar to that observed with heme alone, provides a rationale for further investigation of this compound for eventual use as a supplement to heme therapy of the acute porphyrias and perhaps other conditions in which heme may be of benefit.

Upstream regulatory elements in chick heme oxygenase-1 promoter: A study in primary cultures of chick embryo liver cells

Previously, chick heme oxygenase-1 (cHO-1) gene was cloned by us and two regions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -1573) and a MRE/cMyc complex (-52 to -41). In the current study, the roles of these two elements in mediating the sodium arsenite or cobalt chloride dependent induction of cHO-1 were investigated further. DNA binding studies and site-directed mutagenesis studies indicated that both the AP-1 and MRE/cMyc elements are important for the sodium arsenite induction, while cobalt chloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear proteins binding to the AP-1 element was increased by both sodium arsenite or cobalt chloride treatment, whereas the binding of proteins to the MRE/cMyc element showed a high basal expression in untreated cells and the binding activity was only slightly increased by sodium arsenite treatment. Site-directed mutagenesis studies showed that, to completely abolish sodium arsenite induction, both the AP-1 and MRE/cMyc elements must be mutated; mutation of either element alone resulted in only a partial effect. In contrast, a single mutation at AP-1 element was sufficient to reduce the cobalt chloride induction almost completely. The MRE/cMyc complex plays a major role in the basal level expression, and shares some similarities to the upstream stimulatory factor element (USF) identified in the promoter regions of mammalian HO-1 genes and other stress regulated genes. Because sodium arsenite is known to cause oxidative stress and because activation of AP-1 proteins has been shown to be a key step in the oxidative stress response pathway, we also explored the possibility that the induction of the cHO-1 gene by sodium arsenite is mediated through oxidative stress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction of the endogenous cHO-1 message or cHO-1 reporter construct activities induced by sodium arsenite or cobalt chloride. These antioxidants also reduced the protein binding activities to the AP-1 element in the electrophoretic mobility shift assays. In summary, induction of the cHO-1 gene by sodium arsenite or cobalt chloride is mediated by activation of the AP-1 element located at -1,573 to -1,580 of the 5′ UTR.

Impaired Generation of Hepatitis B Virus-specific Memory B Cells in HIV Infected Individuals Following Vaccination

Hepatitis B-specific memory B cell (HSMBC) frequencies were measured following hepatitis B vaccination in 15 HIV uninfected and 12 HIV infected adolescents. HSMBC were detected at significantly lower frequencies in HIV infected than in HIV uninfected individuals. The detection of HBsAb >10mIU/ml at study week 28 was strongly associated with the detection of HSMBC and a direct correlation between HBsAb titers and HSMBC frequencies was observed. In HIV uninfected individuals, antibody titers >1000mIU/ml were associated with higher HSMC frequencies. Lower HSMBC frequencies, reduced memory B cell (MBC) proliferation, and altered B cell phenotypes were measured in viremic HIV infected individuals compared with aviremic HIV infected or HIV uninfected individuals.

Mechanism of induction of heme oxygenase by metalloporphyrins in primary chick embryo liver cells: evidence against a stress-mediated response.

Heme oxygenase catalyzes the first and rate-controlling step in heme catabolism. One of the two forms of heme oxygenase (heme oxygenase-1) has been shown to be increased by heme, metals, and in some systems, by certain environmental stresses. However, it remains uncertain whether heme induces hepatic heme oxygenase-1 by a general stress response, or a specific heme-dependent cellular response. The work communicated here explores this issue by examining possible mechanisms whereby heme and other metalloporphyrins induce heme oxygenase-1 in normal liver cells. Primary cultures of chick embryo liver cells were tested for their ability to increase heme oxygenase mRNA after exposure to selected metalloporphyrins (heme, chromium mesoporphyrin, cobalt protoporphyrin and manganese protoporphyrin). The ability of antioxidants to decrease metalloporphyrin-mediated induction of heme oxygenase-1 mRNA was also tested. Our results indicate that: 1) the increase in heme oxygenase-1 mRNA mediated by heme or other metalloporphyrins may involve a short-lived protein(s) since the increase was prevented by several inhibitors of protein synthesis; and 2) in normal liver cells, heme-dependent oxidative stress does not play a key role in the heme-mediated induction of heme oxygenase-1. We conclude that heme and other non-heme metalloporphyrins induce heme oxygenase-1 through a mechanism requiring protein synthesis, not because metalloporphyrins increase cellular oxidative or other stress.

Effects of selected antihypertensives and analgesics on hepatic porphyrin accumulation: implications for clinical porphyria.

When patients with acute porphyrias are treated with antihypertensives and analgesics, they could be placed at increased risk of developing porphyric attacks, since little is known about the potential for many of these drugs to induce these attacks. We used primary chick embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of antihypertensives and analgesics on porphyrin accumulation. Cells were treated with desferrioxamine to block heme synthesis partially, simulating conditions encountered in porphyric patients. Typically, cells were treated for 20 hr with the test drugs (3.16 to 1000 microM), along with desferrioxamine. Porphyrins were measured spectrofluorometrically, as uro-, copro,- and protoporphyrin. The evaluated drugs included six antihypertensives (two calcium channel blockers, an angiotensin receptor antagonist, and three inhibitors of angiotensin converting enzyme) and eight analgesics. Of the calcium channel blockers tested, nifedipine greatly increased porphyrin accumulation, whereas diltiazem caused only a slight increase. Losartan (an angiotensin receptor antagonist), captopril, or lisinopril (two angiotensin converting enzyme inhibitors) produced only small increases in porphyrin accumulation. In contrast, enalapril (another angiotensin converting enzyme inhibitor) substantially increased porphyrin accumulation when given in high concentrations. Among the analgesics tested, fentanyl and tramadol produced the highest porphyrin accumulations. Nalbuphine, hydrocodone, oxycodone, and dezocine were moderately or weakly porphyrogenic, whereas buprenorphine and morphine did not increase porphyrin accumulation. These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with nifedipine (compared with diltiazem), enalapril (compared with captopril or lisinopril), and tramadol (compared with the other analgesics).

Induction of heme oxygenase-1 by phenylarsine oxide. Studies in cultured primary liver cells.

Heme oxygenase‐1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular ‘stress’. This has led to the realization that HO‐1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogen‐activated protein kinases and other kinases in the mechanism of induction of HO‐1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO‐1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO‐1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 μM) of PAO produces a marked increase in mRNA and protein of HO‐1. This increase is comparable to the level obtained by addition of heme (20 μM), and occurs without producing changes in cellular glutathione levels or stabilization of HO‐1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO‐1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAO‐mediated induction of HO‐1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO‐1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO‐1 by PAO and by heme may share some common pathways.