|Year : 2020 | Volume
| Issue : 3 | Page : 930-935
p53 as an apoptotic marker before and after phototherapy in hyperbilirubinemic neonates
Gehan K Sead1, Belal A Montaser1, Thoria A Omar1, Zain A Omar2, Amr A Alkorashy1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin El Kom, Menoufia Governorate, Egypt
2 Department of Pediatrics, Faculty of Medicine, Menoufia University, Shebin El Kom, Menoufia Governorate, Egypt
|Date of Submission||11-Jan-2019|
|Date of Decision||24-Feb-2019|
|Date of Acceptance||05-Mar-2019|
|Date of Web Publication||30-Sep-2020|
Belal A Montaser
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Yassin Abd El Ghaffar Street, Shebin El Kom, Menoufia Governorate
Source of Support: None, Conflict of Interest: None
The objective of this study was to study the serum level of p53 before, during, and after phototherapy in full-term neonates with hyperbilirubinemia and to confirm that p53 is an apoptotic marker related to phototherapy.
Jaundice is a serious condition that can lead to death or lifelong neurological sequela in newborns, so it requires medical attention. Phototherapy is the standard treatment for neonatal hyperbilirubinemia; however, it led to potential side effects such as retinal degeneration, diarrhea, dehydration, and skin rash. Experimental studies demonstrated that phototherapy increased apoptosis in lymphoma cell line and neonatal small intestine cells. p53 is a tumor-suppressor protein that regulates the cell cycle preserving stability by preventing genome mutation.
Patients and methods
This study was conducted at the Clinical Pathology and Pediatric Departments of Menoufia University Hospitals from June 2017 to December 2018 on 22 neonates with hyperbilirubinemia necessitating phototherapy, 11 neonates with hyperbilirubinemia not necessitating phototherapy, and 11 apparently healthy normal neonates They have undergone full history, clinical examination, hemoglobin, reticulocyte count, Coombs test, and blood and Rh grouping. Quantification of p53 was done by enzyme-linked immunosorbent assay.
Serum p53 level was highly significant (P < 0.001) in patients with hyperbilirubinemia necessitating phototherapy at the peak of phototherapy compared with other groups.
Serum p53 level was raised in neonates who become exposed to phototherapy and it may be used as a marker of apoptosis.
Keywords: apoptosis, enzyme-linked immunosorbent assay, hyperbilirubinemia, p53, phototherapy
|How to cite this article:|
Sead GK, Montaser BA, Omar TA, Omar ZA, Alkorashy AA. p53 as an apoptotic marker before and after phototherapy in hyperbilirubinemic neonates. Menoufia Med J 2020;33:930-5
|How to cite this URL:|
Sead GK, Montaser BA, Omar TA, Omar ZA, Alkorashy AA. p53 as an apoptotic marker before and after phototherapy in hyperbilirubinemic neonates. Menoufia Med J [serial online] 2020 [cited 2021 Mar 5];33:930-5. Available from: http://www.mmj.eg.net/text.asp?2020/33/3/930/296716
| Introduction|| |
Jaundice is the visible yellow appearance of the skin that occurs with chemical hyperbilirubinemia. It occurs in adults with serum bilirubin of more than 2 mg/dl, and in neonates with serum bilirubin of more than 5 mg/dl. Bilirubin is formed by the breakdown of heme. Newborns have variances in bilirubin production and elimination leading to a rise in bilirubin levels referred to as physiological hyperbilirubinemia that is present in about 60% of all normal neonates in the first days of life . Pathological neonatal hyperbilirubinemia can be caused by increased bilirubin production as in hemolysis or decreased bilirubin clearance such as prematurity. Effective treatments for high bilirubin concentration in infants include intravenous γ-globulin, phototherapy, and exchange transfusion . Neonatal phototherapy (NNPT) is a noninvasive, inexpensive, and safe treatment for neonatal jaundice. It has been used in the management of neonatal unconjugated hyperbilirubinemia for more than half a century. Phototherapy devices include fluorescent, halogen, fiber optic or light-emitting diode light sources . NNPT decreases serum bilirubin levels by converting bilirubin through structural photoisomerization and photooxidation into execrable products. The principal sites of NNPT action are localized in the skin and in capillary circulation under the skin . Although phototherapy is the standard treatment for neonatal hyperbilirubinemia, it may lead to potential side effects such as retinal degeneration, diarrhea, dehydration, and skin rash . This occurs due to initiation of the release of cutaneous reactive nitrogen species and reactive oxygen species and photolysis products are cytotoxic and are associated with the production of free oxygen radicals. Free radicals have been shown to initiate apoptotic cell death in many experimental models . Two major signaling pathways leading to apoptosis have been recognized. First is mitochondria dependent and is controlled by suppressing or inducing genes, such as p53, BCL2, and BAX. Second is death receptor dependent, involving the interaction of death receptors involving Fas/Fasl interaction receptor-associated death proteases and subsequent activation of downstream effector caspases . Apoptosis is a hereditarily controlled mechanism of cell death that is essential for the removal of unwanted cells during normal development and for the maintenance of tissue homeostasis. One of the major apoptosis-signaling pathways involves the p53 tumor suppressor . Tumor protein p53 is a nuclear transcription factor that regulates the expression of a variety of genes involved in apoptosis, growth arrest, or senescence in response to genotoxic or cellular stress. There are four conserved domains in p53: N-terminal domain, which is required for transcriptional transactivation, a sequence-specific DNA-binding domain, a tetramerization domain near the C-terminal end, and a C-terminal domain that interacts directly with single-stranded DNA. Having a short half-life, p53 is normally maintained at low levels in unstressed cells . The aim of the study was toinvestigate the serum level of p53 before, during, and after phototherapy in full-term neonates with hyperbilirubinemia and to confirm that p53 is an apoptotic marker related to phototherapy.
| Patients and Methods|| |
The study was conducted at the Clinical Pathology and Pediatrics Departments of Menoufia University Hospitals from June 2017 to December 2018. It included 44 patients who were classified into three groups:
- Group I (patients): 22 neonates with hyperbilirubinemia necessitating phototherapy (13 males and nine females), their ages ranged from 1 to 3 days
- Group II (control I): 11 neonates with hyperbilirubinemia not necessitating phototherapy (seven males and four females), their ages ranged from 1 to 3 days
- Group III (control II): 11 apparently healthy neonates without hyperbilirubinemia (six males and five females), age-matched and sex-matched to the patient group.
This study is approved by the Research Ethics Committee in Menoufia Faculty of Medicine and informed written consent was taken from parents of every participant in the study. All neonates were submitted to full clinical assessment including prenatal history for pre-existing maternal or fetal problems; natal history for documented history suggestive of prenatal asphyxia, Apgar score; and postnatal history for pulmonary, cardiovascular, or neurological abnormalities; and thorough clinical examination for determination of the gestational age, determination of birth weight, length, and occipitofrontal circumference. Jaundice was checked by Billicheck and compared with standard curves.
Blood sample collection
From all neonates, 2 ml of blood was aseptically collected and was divided into two tubes: one on EDTA for hemoglobin, reticulocytic count, blood and Rh grouping and the other was left to clot for 10–15 min. Serum was separated by centrifugation at 2000 rpm for 10 min. The separated serum was divided into two parts, one for determination of total serum bilirubin (TSB) level (comparing its result with transcutaneous bilirubinometry using Billicheck) and the other was kept frozen at −20°C to be used for measuring p35 as a basal level. Two additional serum samples were taken from patients who received phototherapy, the first just before discontinuation of phototherapy and the second after 72 h of discontinuation of phototherapy for determination of TSB level and p35 level to get rid of any possible toxic or apoptotic effect of phototherapy.
Hemoglobin and reticulocytic count were measured ADVIA 120 Hematology - Siemens Healthineers Globa (Erlangen, Germany). TSB level was measured with Modular AU680 automatic biochemistry Analyzer (Beckman Coulter, Brea, California, USA). Blood group was determined through ORTHO gel cards (Ortho Clinical Diagnostics, Raritan, New Jersey, USA). Coombs test was done on the Matrix gel card (Tulip Diagnostics Private Limited, Gitanjali, Tulip Block, Bambolim, India).
Enzyme-linked immunosorbent assay for p53 measurement
Serum samples were prepared according to the guidelines of the p53 enzyme-linked immunosorbent assay kit. The p53 levels in supernatant serum was evaluated by enzyme-linked immunosorbent assay according to the manufacturer's instructions (Bender Med Systems GmbH, Vienna, Austria).
The results were statistically analyzed by SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA). One-way analysis of variance (ANOVA) (F-test): A one-way analysis of variance is a single test used to collectively indicate the presence of any significant difference between several groups for a normally distributed quantitative variable. Kruskal–Wallis test is the nonparametric version of ANOVA. Repeated measures ANOVA: a single test used to collectively indicate the presence of any significant difference between several time sequences for a normally distributed quantitative variable. Post-hoc test: was used to show any significant difference between individual groups or different time sequences. χ2 is used to compare between two groups or more regarding one qualitative variable. Fisher's exact is used when one of the expected cells is less than 5. Pearson's and Spearman's correlation: to detect the degree and strength of association between variables. Receiver operating characteristic (ROC) curve is a graphical plot of the sensitivity versus false positive rate (1 − specificity). The ROC curve is also known as a relative operating characteristic curve because it is a comparison of two operating characteristics. The difference is considered significant if P value is less than 0.05.
| Results|| |
There were no significant statistical differences between all studied groups regarding demographic data such as age and gender and this means that the groups were well-matched [Table 1].
All biochemical data revealed that there was highly significant statistical differences between the studied groups regarding serum bilirubin level and serum p53 level (P < 0.001), whereas there were no significant statistical differences between all studied groups (P > 0.05) regarding hemoglobin [Table 2].
|Table 2: Mean distribution of laboratory. investigations among the studied groups (baseline study, before phototherapy)|
Click here to view
Regarding reticulocytes count the gained results showed a highly significant statistical difference between all hyperbilirubinemic neonates (either requiring phototherapy or not) and normal neonates' group III with no significant difference between both groups of hyperbilirubinemia.
Post-hoc test showed that there were highly significant statistical differences in p53 between groups I and II, and between groups I and III (P < 0.001) with only significant statistical difference between groups II and III (P = 0.004) [Table 2]. The results of this study showed that the differences regarding bilirubin and p53 between patients of group I at different time sequences were statistically significant (P < 0.001), as the bilirubin level starts at a high level, then declines at the peak of phototherapy and declines more after 72 h of discharge, whereas the p53 level starts at a base level, rise sharply during phototherapy peak, and declines near base levels after 72 h of discharge [Table 3].
|Table 3: Mean distribution bilirubin and p53 among the studied patients at different time sequences|
Click here to view
[Figure 1], [Figure 2] and [Table 4] show the ROC curves regarding p53 between groups I and II and between groups I and III and showing that the best cutoff points of p53 as an apoptotic marker for patient controls and healthy controls are 5.6 and 3.85 U/l, respectively, and at these cutoff points the positive predictive value and negative predictive value for p53 as an apoptotic marker is 100%.
|Figure 1: Receiver operating characteristic (ROC) curve for p53 between patients and patient control I.|
Click here to view
|Figure 2: Receiver operating characteristic (ROC) curve for p53 between patients and healthy control II.|
Click here to view
|Table 4: The best cutoff points of p53 for patients controls and healthy controls|
Click here to view
| Discussion|| |
Unconjugated bilirubin, the principal mammalian bile pigment, is the end product of heme catabolism. Both belong to the superfamily of tetrapyrrolic compounds that serve multiple biological functions . Hyperbilirubinemia is one of the most common conditions opposing neonatologists daily. About 60% of term and 80% of preterm infants develop jaundice in the first days of life. Bilirubin encephalopathy is a devastating brain injury, which can cause permanent neurodevelopmental handicaps . Phototherapy is a noninvasive, easily applicable, and reliable method used widely in the treatment of neonatal jaundice. In general, it has been agreed that the adverse events of phototherapy are not serious and are well-controllable. Nevertheless, unfavorable effects of phototherapy include interruption of the mother–baby relationship, impairment of the circadian rhythm, hypothermia/hyperthermia, and dehydration due to the altered thermal environment of the baby, electrolyte imbalance, bronze baby syndrome, allergic diseases, melanocytic nevus and skin cancers, patent ductus arteriosus, and retinal damage ,. Tumor-suppressor transcription factor p53 is a stress-responsive protein expressed at low levels under common conditions. After stress, p53 is rapidly activated by phosphorylation and interacts with p53-operated response signaling and target genes to form the 'p53 signaling network' to regulate cell cycle arrest, DNA damage repair and induction of cell death. p53 has the ability to temporary block the cell cycle and promote DNA repair. However in certain conditions, p53 is able to induce senescence or promote apoptosis thus providing mechanisms against the accumulation of potentially malignant or defective cells . In this study, the level of p53 was measured before, during, and after phototherapy in full-term neonates with hyperbilirubinemia and to confirm that p53 is an apoptotic marker related to phototherapy. The study evaluated three groups of patients: (a) jaundiced with phototherapy, (b) physiologic jaundice without phototherapy as patient controls, and (c) nonjaundiced completely healthy controls. This approach could stratify the individual impact of phototherapy and jaundice on DNA damage and apoptosis. A full history was taken from all patients and control parents and full clinical examinations were done. In addition to laboratory investigations including TSB level, hemoglobin, blood group, reticulocytic count, and quantification of serum p53 level were done. The three groups were well matched with no statistical difference between them regarding age and sex. All biochemical data has shown that there was a highly significant statistical differences between the studied groups regarding serum bilirubin level and serum p53 level. However, there were no significant statistical differences between all studied groups regarding hemoglobin. In this study, there was significant increase of apoptosis as indicated by a significant increase in p53 induced by phototherapy, whereas hyperbilirubinemia mildly influence p53 levels. Previous studies have evaluated the effect of hyperbilirubinemia and phototherapy on apoptosis. El-Abdin et al.  found that bilirubin and phototherapy significantly induced apoptosis by BCL2 downregulation and increased BAX gene expression. Christensen et al.  found that phototherapy stimulates the release of cutaneous reactive nitrogen species and reactive oxygen species and photolysis products that are cytotoxic and are associated with the production of free oxygen radicals. Free radicals have been shown to initiate apoptotic cell death in many in-vitro and in-vivo experimental models. Regarding reticulocytic count the gained results showed a highly significant statistical difference between hyperbilirubinemic neonates (either requiring phototherapy or not) and normal neonates, whereas there is no significant difference between both groups of hyperbilirubinemia. This means that hyperbilirubinemia had an increasing effect on reticulocytic count but it is still in a reference range for neonates and this could be explained that hyperbilirubinemia may have a stimulating effect on red blood cells precursor production. This agreed with Mesbah-Namin et al.  who stated that jaundice has no negative effects on DNA and on apoptosis induction. In contrast, phototherapy can negatively influence DNA integrity, and induce genotoxic effects and positively stimulate bone marrow erythroid precursors as a response to gene damage . The study results were in agreement with the study of Chotikasemsri et al., who reported that phototherapy increases the expression of cancer-predisposing genes. Mohamed and Niazy  are definite that both intensive and conventional phototherapy may induce genotoxic effect of term infants with hyperbilirubinemia. Tatli et al.  have observed that DNA damage increased significantly with the duration of phototherapy as shown by measurements at 24, 48, and 72 h. However, Aycicek and Erel  did not find any correlation between DNA damage scores and bilirubin levels in neonates receiving phototherapy. Christensen et al., specified that blue light induces single-strand breaks in the DNA of cells in culture in the absence of bilirubin. During irradiation of bilirubin solutions with blue and green phototherapy light, long-lived toxic photoproducts were formed under in-vitro conditions. At high and clinically relevant bilirubin concentrations, the effects of blue and green light were relatively similar.
The results of this study showed that the differences regarding bilirubin and p53 between patients of group I at different time sequences were statistically significant, as the bilirubin level starts at a high level and then declines at the peak of phototherapy and declines more after 72 h of discontinuing phototherapy whereas the p53 level starts at a base level, rise sharply during phototherapy peak, and declines near base levels after 72 h of discontinuing phototherapy. The study also found significant differences regarding bilirubin level between patients group I and patients control II, also between patients group I and healthy control III, and between patients controls group II and healthy controls group III. Regarding p53 level there was a highly significant difference between patients group I and patients controls group II. Also between patients group I and healthy control II and significant difference between patients controls group II and healthy controls group III. This result indicated that hyperbilirubinemia does not influence DNA damage and apoptosis, whereas phototherapy causes DNA damage and induces apoptosis and this result agreed with Yahia et al., who reported that there was a significant increase of apoptosis as indicated by a significant increase in p53 induced by phototherapy; in contrast, hyperbilirubinemia did not influence p53 levels. This result has shown that the best cutoff points of p53 for patient controls and healthy controls were 5.6 and 3.85 U/l, respectively, and at these exact cutoff points the positive predictive value, negative predictive value, sensitivity, specificity, and accuracy of p53 as an apoptotic marker was 100%. Finally, we concluded that p53 is a good apoptotic marker with high sensitivity and specificity. It increased in neonates under phototherapy. Hyperbilirubinemia does not stimulate DNA damage and apoptosis, whereas phototherapy causes DNA damage and induces apoptosis in neonates, so phototherapy should be restricted to those with significant hyperbilirubinemia.
| Conclusion|| |
Serum p53 level was raised in neonates who become exposed to phototherapy and it may be used as a marker of apoptosis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rawat VS. DNA damage induction by bilirubin induced oxidative stress and activation of DNA repair pathways by bilirubin [dissertation]. Milton Keynes, UK: The Open University; 2018.
Ramy N, Ghany A, Alsharany W, Nada A, Darwish R, Rabie A, Aly H. Jaundice, phototherapy and DNA damage in full-term neonates. J Perinatol 2016; 36
Zauk AM. Phototherapy: a simple and safe treatment for neonatal jaundice. J Pediatr Neonatal Care 2015; 2
Xiong T, Qu Y, Cambier S, Mu D. The side effects of phototherapy for neonatal jaundice: what do we know? What should we do? Eur J Pediatr 2011; 170
Gathwala G, Sharma S. Oxidative stress, phototherapy and the neonate. Indian J Pediatr 2000; 67
El-Abdin MYZ, El-Salam MA, Ibrhim MY, Koraa SS, Mahmoud E. Phototherapy and DNA changes in full term neonates with hyperbilirubinemia. Egypt J Med Human Genet 2012; 13
Kahveci H, Dogan H, Karaman A, Caner I, Tastekin A, Ikbal M. Phototherapy causes a transient DNA damage in jaundiced newborns. Drug Chem Toxicol 2013; 36
Bruzell Roll E. Bilirubin-induced cell death during continuous and intermittent phototherapy and in the dark. Acta Paediatr 2005; 94
Kara S, Yalniz-Akkaya Z, Yeniaras A Ornek F, Bilge YD. Ocular findings on follow-up in children who received phototherapy for neonatal jaundice. J Chinese Med Assoc 2017; 80
Giovannelli L, Pitozzi V, Riolo S, Dolara P. Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay. Mutat Res 2003; 538
Lia X, Yub Q, Tana W, Zhanga Z, Ma H. MicroRNA-125b mimic inhibits ischemia reperfusion-induced neuroinflammation and aberrant p53 apoptotic signaling activation through targeting TP53INP1. Brain Behav Immun 2018; 74
Christensen T, Reitan JB, Kinn G. Single-strand breaks in the DNA of human cells exposed to visible light from phototherapy lamps in the presence and absence of bilirubin. J Photochem Photobiol B 1990; 7
Mesbah-Namin SA, Shahidi M, Nakhshab M. An increased genotoxic risk in lymphocytes from phototherapy-treated hyperbilirubinemic neonates. Iran Biomed J 2017; 21
Chotikasemsri P, Tangtrakulwanich B, Sangkhathat S. The effect of phototherapy on cancer predisposition genes of diabetic and normal human skin fibroblasts. Biomed Res Int 2017; 2017
Mohamed WW, Niazy WH. Genotoxic effect of phototherapy in term newborn infants with hyperbilirubinemia. J Neonatal Perinatal Med 2012; 5
Tatli MM, Minnet C, Kocyigit A, Karadag A. Phototherapy increases DNA damage in lymphocytes of hyperbilrubinemic neonates. Mutat Res 2008; 654
Aycicek A, Erel O. Total oxidant/antioxidant status in jaundiced newborns before and after phototherapy. J Pediatr (Rio J) 2007; 83
Yahia S, Shabaan AE, Gouida M, El-Ghanam D, Eldegla H, Amal El-Bakary A, Hesham Abdel-Hady H. Influence of hyperbilirubinemia and phototherapy on markers of genotoxicity and apoptosis in full-term infants. Eur J Pediatr 2014; 174
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]