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Heme Degradation by Heme Oxygenase Protects Mitochondria but Induces ER Stress via Formed Bilirubin.

Müllebner A, Moldzio R, Redl H, Kozlov AV, Duvigneau JC - Biomolecules (2015)

Bottom Line: The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress.The mechanisms by which HO mediates the described effects are not completely clear.However, the proteins that are targeted by BR still have to be identified.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Biochemistry, Veterinary University Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. andrea.muellebner@vetmeduni.ac.at.

ABSTRACT
Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.

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BR results in decreased proliferation and metabolic activity of BRL 3A cells. (A) Cells were treated with vehicle (Veh) or BR in the concentrations indicated, and incubated for 48 h. Cell number (squares and dotted line) was determined using crystal violet assay, and expressed in % relative to the vehicle control (CV 48 h). Data are given as means (±SD) obtained from two experiments with n = 2 replicates. Metabolic activity of treated cells was determined by MTT assay (diamonds and full line). Data are given as means (±SD) obtained from one experiment with n = 4 replicates (MTT 48 h). Significant differences to the control are indicated as (*, §); (B) Determination of cell numbers within one experiment at consecutive time points (12 h and 48 h) showed a decreased proliferation rate in the presence of BR. Data are given as % increase relative to the values determined at 12 h.
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biomolecules-05-00679-f007: BR results in decreased proliferation and metabolic activity of BRL 3A cells. (A) Cells were treated with vehicle (Veh) or BR in the concentrations indicated, and incubated for 48 h. Cell number (squares and dotted line) was determined using crystal violet assay, and expressed in % relative to the vehicle control (CV 48 h). Data are given as means (±SD) obtained from two experiments with n = 2 replicates. Metabolic activity of treated cells was determined by MTT assay (diamonds and full line). Data are given as means (±SD) obtained from one experiment with n = 4 replicates (MTT 48 h). Significant differences to the control are indicated as (*, §); (B) Determination of cell numbers within one experiment at consecutive time points (12 h and 48 h) showed a decreased proliferation rate in the presence of BR. Data are given as % increase relative to the values determined at 12 h.

Mentions: To elucidate the role of BR in regulating proliferation of BRL3A cells we incubated cells with varying concentrations of unconjugated BR and determined cell number (crystal violet assay; [38,39,40]) and metabolic activity (MTT assay; [41]) at different time points (12 h and 48 h; Figure 7). Considering that an increase in HO would only slowly increase physiologic levels of BR, we used physiologic concentrations of BR, which range between 5 and 32 µM in human serum [42] and about half as much in rodents [43], of which around 4% appears as water-soluble glucuronides [44]. We found that physiologic levels of BR (4–20 µM) decreased the proliferation rate about 20%, but affected the metabolic activity to a much higher degree (50% activity after 48h at the highest BR concentration tested).


Heme Degradation by Heme Oxygenase Protects Mitochondria but Induces ER Stress via Formed Bilirubin.

Müllebner A, Moldzio R, Redl H, Kozlov AV, Duvigneau JC - Biomolecules (2015)

BR results in decreased proliferation and metabolic activity of BRL 3A cells. (A) Cells were treated with vehicle (Veh) or BR in the concentrations indicated, and incubated for 48 h. Cell number (squares and dotted line) was determined using crystal violet assay, and expressed in % relative to the vehicle control (CV 48 h). Data are given as means (±SD) obtained from two experiments with n = 2 replicates. Metabolic activity of treated cells was determined by MTT assay (diamonds and full line). Data are given as means (±SD) obtained from one experiment with n = 4 replicates (MTT 48 h). Significant differences to the control are indicated as (*, §); (B) Determination of cell numbers within one experiment at consecutive time points (12 h and 48 h) showed a decreased proliferation rate in the presence of BR. Data are given as % increase relative to the values determined at 12 h.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496691&req=5

biomolecules-05-00679-f007: BR results in decreased proliferation and metabolic activity of BRL 3A cells. (A) Cells were treated with vehicle (Veh) or BR in the concentrations indicated, and incubated for 48 h. Cell number (squares and dotted line) was determined using crystal violet assay, and expressed in % relative to the vehicle control (CV 48 h). Data are given as means (±SD) obtained from two experiments with n = 2 replicates. Metabolic activity of treated cells was determined by MTT assay (diamonds and full line). Data are given as means (±SD) obtained from one experiment with n = 4 replicates (MTT 48 h). Significant differences to the control are indicated as (*, §); (B) Determination of cell numbers within one experiment at consecutive time points (12 h and 48 h) showed a decreased proliferation rate in the presence of BR. Data are given as % increase relative to the values determined at 12 h.
Mentions: To elucidate the role of BR in regulating proliferation of BRL3A cells we incubated cells with varying concentrations of unconjugated BR and determined cell number (crystal violet assay; [38,39,40]) and metabolic activity (MTT assay; [41]) at different time points (12 h and 48 h; Figure 7). Considering that an increase in HO would only slowly increase physiologic levels of BR, we used physiologic concentrations of BR, which range between 5 and 32 µM in human serum [42] and about half as much in rodents [43], of which around 4% appears as water-soluble glucuronides [44]. We found that physiologic levels of BR (4–20 µM) decreased the proliferation rate about 20%, but affected the metabolic activity to a much higher degree (50% activity after 48h at the highest BR concentration tested).

Bottom Line: The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress.The mechanisms by which HO mediates the described effects are not completely clear.However, the proteins that are targeted by BR still have to be identified.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Biochemistry, Veterinary University Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. andrea.muellebner@vetmeduni.ac.at.

ABSTRACT
Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.

Show MeSH
Related in: MedlinePlus