key: cord-0030173-mh4eb8w0
authors: Ma, Christopher D.; Van Horn, Cynthia G.; Wan, Meimei; Bishop, Colin; Bonkovsky, Herbert L.
title: Assessment of porphyrogenicity of drugs and chemicals in selected hepatic cell culture models through a fluorescence‐based screening assay
date: 2022-04-21
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.951
sha: 027232b4bde2ae39df5a79ffd5d390e03e76586d
doc_id: 30173
cord_uid: mh4eb8w0

Compounds that induce 5‐aminolevulinic acid [ALA] synthase‐1 and/or cytochromes P‐450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence‐based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase‐1 [ALAS‐1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS‐1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, β‐estradiol, hydroxychloroquine sulfate, alpha‐methyldopa, D (‐) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi‐well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

Hepatic 5-aminolevulinic acid synthase-1 [ALAS -1] , an enzyme that converts glycine and succinyl coenzyme A into 5-aminolevulinic acid [ALA] , is a tightly regulated, rate-determining enzyme in the heme biosynthesis pathway. 1, 2 Conditions, including hypoxia and oxidative stress, and compounds, such as alcohol, barbiturates, diverse xenobiotics, progesterone, and hydantoins, may induce ALAS-1 activity and upregulate cytochromes P450, stimulating the production of the end product, heme. [2] [3] [4] [5] [6] The size of the regulatory heme pool in hepatocytes plays a major role in ALAS-1 regulation by decreasing transcription of the ALAS-1 gene, reducing the rate of transport of ALAS-1 into the mitochondria at higher concentrations, upregulating ALAS-1 at lower concentrations, and increasing the breakdown of the mature enzyme in mitochondria by the action of LONP1 protease. [7] [8] [9] [10] [11] [12] [13] If a step distal to that of ALAS-1 is defective in the heme biosynthesis pathway, for example, due to an inherited or acquired enzyme deficiency, a relative deficit in a small but critical regulatory heme pool may occur, leading to marked upregulation of ALAS-1 and overproduction of ALA. This overproduction is thought to be the major cause of acute neurovisceral attacks, autonomic abnormalities, and other neuromuscular features, which are hallmarks of acute porphyric attacks. 2, 3, 14 Patients with acute hepatic porphyrias [AHPs], including ALA dehydratase deficiency porphyria, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, may experience acute porphyric attacks with marked induction of ALAS-1. 15 Thus, it is important and clinically relevant to test for porphyrogenicity of common and novel medications to determine whether they have the potential to trigger or exacerbate acute porphyric attacks in patients with the AHPs.

Currently, there is no standard accepted model to assess for porphyrogenicity of drugs or other chemicals. Historically, a standard of cell models in human hepatology has been the utilization of primary human hepatocytes [PHHs] ; however, due to many limitations to the use of PHH, such as rapid de-differentiation and slow proliferation in vitro, limited supply, and high costs, alternative cell culture models have been developed and tested with the general goal of providing a physiologically relevant model system. 16 Two notable hepatic cell lines with ALAS-1 inducibility are LMH cells, which are a hepatocellular carcinoma cell line that arose in a male leghorn chicken treated with diethylnitrosamine, and HepG2 cells, which are a human hepatoma cell line originally derived from a hepatocellular carcinoma that arose in a 15-year-old boy with chronic hepatitis B infection. 10, [17] [18] [19] [20] [21] Similarly to heme synthesis in humans, ALAS-1 transcription activity in LMH cells is mediated by heme-dependent regulation, which can repress both drug-mediated and basal induction of ALAS-1, supporting the fact that, although LMH cells are not derived from human hepatocytes, they are relevant to human heme biosynthesis. 10 There have been several reports of the use of LMH and HepG2 cells to study porphyrogenicity,however, it is unclear which cell line has retained the capacity for upregulation of ALAS-1 and for porphyrin accumulation to facilitate facile detection and quantification via fluorospectrometry. 10, 21, 22 3D hepatic spheroids have recently been emerging as newer models to study drug-induced liver injury-related pathologies and liver toxicology studies purported to exhibit higher sensitivity and specificity in comparison to 2D models. 23 27, 28 This also made it possible to have a greater accumulation of fluorescent PP, improving the sensitivity of a high-throughput, microplate-based assay procedure. 28 The addition of ALA, which bypasses the need for upregulation of ALAS-1, provides information regarding the functional status of the biosynthetic pathway from ALA to PP in each cell model. Acetaminophen and acetylsalicylic acid, which have been used without causing acute porphyric attacks or upregulation of ALAS-1 in patients for decades, served as negative controls. β-estradiol, alpha-methyldopa, D (-) norgestrel, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan were also tested as they have been hypothesized to induce simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

cytochromes P-450, delta-[or 5-] aminolevulinic acid [synthase], heme, liver cell cultures, liver cell organoids, porphyria, porphyrins acute porphyric attacks. Hydroxychloroquine sulfate was tested as well because, while we were completing this study, this compound gained significant popularity due to its claimed benefits for treating COVID-19 infections and because our patients with AHP asked about its safety.

The HepG2 cells were supplied by ATCC and maintained in DMEM and endothelial cells at 80%, 10%, 5%, and 5%, respectively. 23, 26 All cell lines were incubated in a 37°C and 5% CO 2 incubator. 

LMH cells were seeded in 0.1% gelatin-coated, 60-mm dishes (4 × 10 6 cells per plate) and were incubated overnight at 37°C. Eppendorf tube and the pH was adjusted to between 3 and 5 using 4-M NaOH and pH paper. 29

The conditions for HPLC of porphyrins were essential as described in Ref. [29] .

Results are presented as mean values ± SEM (n = 3). Data were analyzed with the GraphPad Prism 8.0 software. Statistical analyses were calculated by comparing the fluorescence ratios between the negative and positive controls in the presence of DFO to the ratios between the negative control and the drug concentration that produced the greatest fluorescence reading in the presence of DFO. If the latter ratio is greater than the former, then porphyrogenicity is indicated at that concentration for the given compound. A two-sided Student's t-test was used to assess the statistical significance of the two ratios. Figure S3 ).

HepG2 cells at the concentrations tested ( Figure S4 ). Regardless of the expected results, all fluorescence readings, except for the ones produced by ALA, were near the lower detection limit of the fluorospectrometer used, limiting the usefulness of the assay in this cell line. 

In this work, we found that LMH cells have the most robust inducible Recently, liver organoids have been shown to have utility in liver toxicology studies due to the organoids' promising properties to retain normal hepatic characteristics, mimicking in vivo environments. 24, 25, 30 There are still many applications that have not heretofore been reported, such as screening for porphyrogenicity and diseases involving fibrosis and hepatitis, due to multiple limitations, including genomic instability, expression of immature or fetal markers, and limited cell maturation. 31, 32 Our results show that HepG2 organoids and human liver organoids do retain the ability to convert ALA to PP, however, under the conditions of our studies, neither organoid system showed any inducibility of ALAS-1, which is the key biochemical hallmark that underlies porphyrogenicity and drug risk in the AHPs. Perhaps, more complicated systems, such as perfused "liver on a chip" may prove to retain such inducibility. 33 Fluorescence readings from the organoid studies might, perhaps, be increased if the cells were treated for a longer period of time, but this would reduce the practicability of the system for use in a rapid, screening assay. The main goal of this assay is to develop a quick and affordable method to detect porphyrogenicity, thus there is no much-added benefit from increasing the length of incubation just to obtain higher fluorescence readings, especially when compared with the results from the LMH cells.

In the heme biosynthesis pathway, uroporphyrin, coproporphyrin, and PP are maximally fluorescent at somewhat different wavelengths in the red part of the visible light spectrum, but in most cell types exposed to ALA or with induction of ALAS-1, PP is the principal porphyrin that accumulates. This is especially true when DFO is present, which prevents the conversion of PP to heme by chelating iron, which is required for the activity of ferrochelatase. 27, 28, 34, 35 This was confirmed after quantification of these intermediates via HPLC after ALA and AIA treatment in LMH cells ( Figure S5 ). PP was in greater abundance than the other porphyrins [8-to 3-carboxyl-porphyrins].

The complexities of biological in vivo models make it challenging to interpret results from in vitro assays, such as this in vitro porphyrogenic drug screening assay, and to translate them for clinical use.

Currently, in the clinical drug development process, pharmacokinetic and pharmacodynamic studies are required for the three phases of drug approval. 36 Since thorough and rigorous studies are needed to be completed for novel drugs before they can enter the market, β-estradiol and norgestrel were used to represent the estrogen and progestogen classes, respectively, and we found that, at higher concentrations, both led to a notable accumulation of PP, especially in the presence of DFO (Figure 2) . Norgestrel, however, produced greater fluorescence readings than β-estradiol, which is in keeping with clinical impressions and observations in human subjects. 2, [47] [48] [49] [50] [51] [52] We also tested alpha-methyldopa because it continues often to be used to control systemic arterial hypertension in pregnant women.

Although the drug had a low CC 50 of 0.164 mM in the presence of DFO, it did not lead to an appreciable accumulation of PP below concentrations that led to appreciable cytotoxicity. We also found that valsartan, another widely used antihypertensive, did not show evidence of porphyrogenicity. This is reassuring for clinicians treating patients with AHP, in whom systemic arterial hypertension is common, especially during acute attacks, and often continuing between attacks. These results extend results from earlier studies in our laboratory, which also showed the safety of losartan, captopril, and lisinopril. 35 We also tested hydroxychloroquine because, while we were doing this study, this compound gained significant popularity, for a time, due to its claimed benefits for treating COVID-19 infections.

Our results indicate that hydroxychloroquine does not upregulate hepatic ALAS-1 and is not risky for patients with AHPs; however, it did show cytotoxicity with a CC 50 of 0.381 mM in the presence of DFO. We also note that, in recent studies, hydroxychloroquine had no added benefit in treating COVID-19. [53] [54] [55] Sulfonamide-containing antibiotics, such as sulfamethoxazole and sulfisoxazole, have been considered to be risky for use in pa- although it does list the combination of trimethoprim and sulfamethoxazole [Bactrim] as porphyrogenic, suggesting that the agent of concern is more likely trimethoprim. Thus, based on our results and our review of published literature, we are unconvinced that either sulfisoxazole or sulfamethoxazole without trimethoprim is truly risky for use in patients with AHPs.

In summary, among several in vitro model systems tested, the LMH cell model was the best system to predict the porphyrogenicity of various drugs in our fluorescence-based in vitro drug screening assay. The positive controls, AIA, ALA, phenobarbital, and phenytoin, showed significant accumulation of protoporphyrin, as expected, and the negative controls, acetaminophen and acetylsalicylic acid, showed significantly less effect. Norgestrel, phenobarbital, phenytoin, and sodium valproate all produced fluorescence readings higher than the AIA control at concentrations above 0.314 mM, which indicates that these compounds may likely induce or exacerbate acute porphyric attacks. Pharmacodynamic and pharmacokinetic studies and years of clinical observations support this conclusion; these drugs should best be avoided by patients with AHP. Since such studies are required in the drug approval process and as more novel medications enter the market with the unknown potential to exacerbate the hepatic porphyrias, it would be ideal for the quick and inexpensive assay herein described to be included in the batteries of tests now routinely performed for new candidate drugs. Patients with AHP and their health-care providers would benefit from such knowledge. 

This study is exempt from ethics approval.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

https://orcid.org/0000-0002-4520-5849

Herbert L. Bonkovsky https://orcid.org/0000-0001-7387-3230

A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues

Porphyrin and heme metabolism and the porphyrias

Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress

Effect of alcohol on the heme and porphyrin synthesis interaction with phenobarbital and pyrazole

Carbamazepine in seizure management in acute intermittent porphyria

Hepatic 5-aminolevulinic acid synthase mRNA stability is modulated by inhibitors of heme biosynthesis and by metalloporphyrins

Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal

Heme regulates hepatic 5-aminolevulinate synthase mRNA expression by decreasing mRNA half-life and not by altering its rate of transcription

Haem repression of the housekeeping 5-aminolaevulinic acid synthase gene in the hepatoma cell line LMH

Regulation by heme of mitochondrial protein transport through a conserved amino acid motif

5-aminolevulinic acid (ALA) deficiency causes impaired glucose tolerance and insulin resistance coincident with an attenuation of mitochondrial function in aged mice

Lon peptidase 1 (LONP1)-dependent breakdown of mitochondrial 5-aminolevulinic acid synthase protein by heme in human liver cells

Phase 3 Trial of RNAi therapeutic givosiran for acute intermittent porphyria

Ala Dehydratase Deficiency Porphyria

The breakthrough in primary human hepatocytes in vitro expansion

Culture and functional characterization of human hepatoma HepG2 cells

Drugs mediate the transcriptional activation of the 5-aminolevulinic acid synthase (ALAS1) gene via the chicken xenobiotic-sensing nuclear receptor (CXR)

Delta-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals

Establishment and characterization of a chicken hepatocellular carcinoma cell line

Heme biosynthesis in a chicken hepatoma cell line (LMH): comparison with primary chick embryo liver cells (CELC)

Repression of ALA synthase by heme and zinc-mesoporphyrin in a chick embryo liver cell culture model of acute porphyria

HepG2 (C3A) spheroids show higher sensitivity compared to HepaRG spheroids for drug-induced liver injury (DILI)

Developing a toolbox for drug-induced liver injury

High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

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

Ferrochelatase deficiency abrogated the enhancement of aminolevulinic acid-mediated protoporphyrin IX by iron chelator deferoxamine

High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials

Use of 3D human liver organoids to predict drug-induced phospholipidosis

Organoids to model liver disease

Liver organoids: from basic research to therapeutic applications

Reproducing human and cross-species drug toxicities using a liver-chip

Effects of antidepressants and benzodiazepinetype anxiolytic agents on hepatic porphyrin accumulation in primary cultures of chick embryo liver cells

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

The use of mechanistic evidence in drug approval

Drug treatment in acute porphyria

Pharmacokinetics and pharmacodynamics of acetylsalicylic acid after intravenous and oral administration to healthy volunteers

Pharmacokinetics and bioavailability study on acetaminophen oral drop in healthy volunteers

The influence of allyl isopropyl acetamide on d-aminolevulinic acid synthetase and cytochrome P-450

Influence of allylisopropylacetamide and phenobarbital treatment on in vivo antipyrine metabolite formation in rats

Phenobarbital pharmacokinetics and bioavailability in adults

Bioavailability of phenytoin sodium capsules available in Thailand. Part II: in vivo study

Safety and tolerability of intravenous valproic acid in healthy subjects: a phase I doseescalation trial

Pharmacokinetics of levonorgestrel 0.75 mg tablets

Acute intermittent porphyria in women: clinical expression, use and experience of exogenous sex hormones. A population-based study in northern Sweden

Uncontrolled acute intermittent porphyria as a cause of spontaneous abortion

Effect of menstrual cycle on acute intermittent porphyria

Effect of estradiol on the induction of porphyria by hexachlorobenzene in the rat

Hereditary uroporphyrinogendecarboxylase deficiency predisposing porphyria cutanea tarda (chronic hepatic porphyria) in females after oral contraceptive medication

Update review of the acute porphyrias

A randomized trial of hydroxychloroquine as postexposure prophylaxis for Covid-19

Effect of hydroxychloroquine with or without azithromycin on the mortality of coronavirus disease 2019 (COVID-19) patients: a systematic review and meta-analysis

A clusterrandomized trial of hydroxychloroquine for prevention of Covid-19

The effect of therapeutic drugs and other pharmacologic agents on activity of porphobilinogen deaminase, the enzyme that is deficient in intermittent acute porphyria