Phototherapy increases DNA damage in lymphocytes of hyperbilirubinemic neonates

Phototherapy is commonly used in the treatment of hyperbilirubinemia in newborns. No serious side effects related to phototherapy have been observed, but concerns regarding its potential to damage DNA have been expressed, based on animal or cell-culture studies. The aim of this study was to investigate, in neonates with hyperbilirubinemia, the possible relation between phototherapy and DNA damage. The study included 33 full-term newborns with non-physiological jaundice and 14 healthy newborns with physiological jaundice as controls. Phototherapy was performed with an array of six fluorescent lamps producing radiation with wavelengths of 480-520 nm at 12 microW/cm(2)/nm. DNA damage in lymphocytes was determined by use of the alkaline comet assay. The DNA damage increased significantly with the duration of phototherapy, as shown by measurements at 24, 48, and 72 h (P<0.001). These findings indicate that phototherapy, widely used in neonatology units, increases DNA damage in newborns. It remains to be seen whether the genotoxic effect observed in the present study can cause any long-term health effect in phototherapy-treated infants in later life.     

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1^−/− mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1^−/− mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1^−/− but not in C57BL/6-Ugt1^−/− mice. Survival of FVB/NJ-Ugt1^−/− mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1^−/− mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0–P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1^−/− mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1^−/− mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions.KEY WORDS: Neonatal jaundice, Ugt1, Phototherapy, BIND, Mouse model     

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb²⁺) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 μM to 200 μM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 μM Pb²⁺. In tobacco plants growing for 6 weeks in soil polluted with Pb²⁺ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb²⁺-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb²⁺, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb²⁺ accumulation in the above-ground parts may explain the lower levels or the absence of Pb²⁺-induced DNA damage in leaves.     

DNA protective properties of vanillin against γ-radiation under different conditions: Possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of γ-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to γ-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of γ-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, ‘tail moment’ and ‘Olive tail moment’. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to γ-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS⁺ radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against γ-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

The UVB-induced synthesis of vitamin D3 and 1α,25-dihydroxyvitamin D3 (calcitriol) in organotypic cultures of keratinocytes: Effectiveness of the narrowband Philips TL-01 lamp (311 nm)

Both calcitriol and UVB radiation exert potent antipsoriatic effects. We hypothesize that the therapeutical effect of UVB radiation may be attributed at least in part to UVB-triggered cutaneous synthesis of calcitriol. The optimum wavelength for initiation of the vitamin D₃ pathway was found to be in the range of 300 ± 5 nm in vitro and in vivo. The narrowband Philips TL-01 lamp which is commonly used as UVB source for phototherapy of psoriasis has maximum spectral irradiance at around 311 nm which is presumed to be, however, of lesser importance in photochemical activation of the vitamin D₃ pathway. The aim of this study was to compare the vitamin D₃ and calcitriol-inducing potential of UVB from the TL-01 lamp with that of monochromatic UVB at 300 ± 2.5 nm and 310 ± 2.5 nm in organotypic cultures of keratinocytes supplemented with 25 μM 7-DHC. We found that maximum calcitriol-generating capacity of the TL-01 lamp at 500 mJ/cm² and 16 h after irradiation still amounts up to 44% of that found after monochromatic irradiation at 300 ± 2.5 nm and 30 mJ/cm². Thus, the antipsoriatic effect of UVB emitted from the TL-01 lamp may, at least in part, based on the antiproliferative and prodifferentiative action of newly synthesized calcitriol on epidermal keratinocytes.     

Effect of phototherapy on the frequency of the use of exchange transfusion in the treatment of neonatal jaundice

The authors analysed an incidence of performed exchange blood transfusions at the newborn babies ward prior to and after introduction of phototherapy into practice. This analysis included the causes of jaundice in newborn. The study included the causes of jaundice in newborn. The study involved 8,937 newborn babies delivered between 1981 and 1985. Prior to phototherapy (period between January, 1981 and July, 1980), 45 blood transfusions and 9 retransfusions were performed. During the period II (between July, 1983 and December, 1985), i.e. phototherapy, 30 blood transfusions and 1 retransfusion were effected despite of the higher number of delivered babies. The obtained results have shown favourable effect of the phototherapy in jaundice of perinatal period, especially in jaundice unconnected with blood Rh factor conflict and in premature babies. Phototherapy decreased the number of performed transfusions and retransfusions.     

1,2,3-Triazole Fused Quinoline-Peptidomimetics: Studies on Synthesis, DNA Binding and Photonuclease Activity

Abstract  

We present, simple approach for the accession of 1,2,3-triazole fused quinoline peptide analogues from 3-(azidomethyl)-2-chloroquinoline in a three-step mechanistic pathway. The UV–Visible absorbance plot shows dynamic interaction of parent triazole derivative with CT DNA as efficient DNA intercalator (K _b = 4.6 × 10⁻⁴ M⁻¹). Finally, the efficient DNA damage was observed on photo-irradiation at 360 nm in the presence of 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-propionic acid 1-(2-chloro-quinolin-3-ylmethyl)-1H-[1,2,3]triazole-4-ylmethyl ester (6a).     

Clinical implications of sperm DNA damage in IVF and ICSI: updated systematic review and meta-analysis

The clinical effect of sperm DNA damage in assisted reproduction has been a controversial topic during recent decades, leading to a variety of clinical practice recommendations. While the latest European Society of Human Reproduction and Embryology (ESHRE) position report concluded that DNA damage negatively affects assisted reproduction outcomes, the Practice Committee of the American Society for Reproductive Medicine (ASRM) does not recommend the routine testing of DNA damage for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Herein, our aim was to perform a systematic review and meta-analysis of studies investigating whether sperm DNA damage affects clinical outcomes in IVF and ICSI, in order to contribute objectively to a consistent clinical recommendation. A comprehensive systematic search was conducted according to PRISMA guidelines from the earliest available online indexing year until March 2020, using the MEDLINE-PubMed and EMBASE databases. We included studies analysing IVF and/or ICSI treatments performed in infertile couples in which sperm DNA damage was well defined and assessed. Studies also had to include information about pregnancy, implantation or live birth rates as primary outcomes. The NHLBI-NIH quality assessment tool was used to assess the quality of each study. Meta-analyses were conducted using the Mantel–Haenszel method with random-effects models to evaluate the Risk Ratio (RR) between high-DNA-damage and control groups, taking into account the 95% confidence intervals. Heterogeneity among studies was evaluated using the I² statistic. We also conducted sensitivity analyses and post-hoc subgroup analyses according to different DNA fragmentation assessment techniques. We identified 78 articles that met our inclusion and quality criteria and were included in the qualitative analysis, representing a total of 25639 IVF/ICSI cycles. Of these, 32 articles had sufficient data to be included in the meta-analysis, comprising 12380 IVF/ICSI cycles. Meta-analysis revealed that, considering IVF and ICSI results together, implantation rate (RR = 0.74; 95% CI = 0.61–0.91; I² = 69) and pregnancy rate (RR = 0.83; 0.73–0.94; I² = 58) are negatively influenced by sperm DNA damage, although after adjustment for publication bias the relationship for pregnancy rate was no longer significant. The results showed a non-significant but detrimental tendency (RR = 0.78; 0.58–1.06; I² = 72) on live birth rate. Meta-analysis also showed that IVF outcomes are negatively influenced by sperm DNA damage, with a statistically significant impact on implantation (RR = 0.68; 0.52–0.89; I² = 50) and pregnancy rates (RR = 0.72; 0.55–0.95; I² = 72), although the latter was no longer significant after correction for publication bias. While it did not quite meet our threshold for significance, a negative trend was also observed for live birth rate (RR = 0.48; 0.22–1.02; I² = 79). In the case of ICSI, non-significant trends were observed for implantation (RR = 0.79; 0.60–1.04; I² = 72) or pregnancy rates (RR = 0.89; 0.78–1.02; I² = 44), and live birth rate (RR = 0.92; 0.67–1.27; I² = 70). The current review provides the largest evidence to date supporting a negative association between sperm DNA damage and conventional IVF treatments, significantly reducing implantation and pregnancy rates. The routine use of sperm DNA testing is therefore justified, since it may help improve the outcomes of IVF treatments and/or allow a given couple to be advised on the most suitable treatment. Further well-designed controlled studies on a larger number of patients are required to allow us to reach more precise conclusions, especially in the case of ICSI treatments.     

Nitric oxide production in newborns under phototherapy.

Nitrogen monoxide (NO) is a potent endogenous vasodilator and is involved in cytotoxicity, neurotransmission, and immunological defense mechanisms. Phototherapy has long been known to change the distribution of blood flow throughout the body in newborn infants. The objective of this study was to investigate the effect of phototherapy on NO production in otherwise healthy newborns. Urinary NO levels were measured before and 6 h after phototherapy by a chemiluminescence method using Sievers NOA. Ten newborns (gestational age, 36.4 +/- 3.9 weeks; birth weight, 2863 +/- 677.44 g; postnatal age, 5.1 +/- 2.72 days) were started on phototherapy according to AAP guidelines and urine for NO measurement was collected prior to therapy and 6 h after the commencement of treatment. Urinary NO levels measured during phototherapy were significantly higher (108.8+/-50.69 micromol/mmol creatinine) than the levels measured before phototherapy (73.13+/-34.15 micromol/mmol creatinine; P < 0.05). These results suggest that newborns receiving phototherapy might have increased NO production, which might result in hemodynamic changes. However, further studies on the effects of phototherapy on NO and photorelaxation are needed before reaching firm conclusions.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel^® (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0–500.0 μg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r = 0.48; P > 0.05) nor for the commercial formulation (r = 0.58, P > 0.05). For the 200.0 μg/ml and 500.0 μg/ml dicamba doses and the 500.0 μg/ml banvel^® dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r = −0.98, P < 0.05) or banvel^® (r = −0.88, P < 0.01) titrated into cultures in the 1.0–500.0 μg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel^® within a 50.0–500.0 μg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P < 0.01); concomitantly, a decrease of undamaged cells was found over control values (P < 0.01). In banvel^®-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P < 0.01) regardless of its concentration whereas banvel^® induced the same effect only within 100.0–500.0 μg/ml dose range (P < 0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel^® to induce DNA and cellular damage on CHO cells.     

Metformin does not prevent DNA damage in lymphocytes despite its antioxidant properties against cumene hydroperoxide-induced oxidative stress

Metformin (1-(diaminomethylidene)-3,3-dimethyl-guanidine), which is the most commonly prescribed oral antihyperglycaemic drug in the world, was reported to have several antioxidant properties such as the inhibition of advanced glycation end-products. In addition to its use in the treatment of diabetes, it has been suggested that metformin may be a promising anti-aging agent. The present work was aimed at assessing the possible protective effects of metformin against DNA-damage induction by oxidative stress in vitro. The effects of metformin were compared with those of N-acetylcysteine (NAC). For this purpose, peripheral blood lymphocytes from aged (n = 10) and young (n = 10) individuals were pre-incubated with various concentrations of metformin (10–50 μM), followed by incubation with 15 μM cumene hydroperoxide (CumOOH) for 48 h, under conditions of low oxidant level, which do not induce cell death. Protection against oxidative DNA damage was evaluated by use of the Comet assay and the cytokinesis-block micronucleus technique. Changes in the levels of malondialdehyde + 4-hydroxy-alkenals, an index of oxidative stress, were also measured in lymphocytes. At concentrations ranging from 10 μM to 50 μM, metformin did not protect the lymphocytes from DNA damage, while 50 μM NAC possessed an effective protective effect against CumOOH-induced DNA damage. Furthermore, NAC, but not metformin, inhibited DNA fragmentation induced by CumOOH. In contrast to the lack of protection against oxidative damage in lymphocyte cultures, metformin significantly protected the cells from lipid peroxidation in both age groups, although not as effective as NAC in preventing the peroxidative damage at the highest doses. Within the limitations of this study, the results indicate that pharmacological concentrations of metformin are unable to protect against DNA damage induced by a pro-oxidant stimulus in cultured human lymphocytes, despite its antioxidant properties.     

Genotoxic effects of PCB 52 and PCB 77 on cultured human peripheral lymphocytes

Polychlorinated biphenyls (PCBs) are known to be carcinogenic, but the mechanisms of this action are uncertain. Most, but not all, studies have concluded that PCBs are not directly mutagenic, and that much if not all of the carcinogenic activity resides in the fraction of the PCB mixture that contains congeners with dioxin-like activity. The present study was designed to determine genotoxic effects of an ortho-substituted, non-coplanar congener, 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), and a non-ortho-substituted coplanar congener with dioxin-like activity, 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) on cultured human peripheral lymphocytes. DNA damage was assessed by use of the comet assay (alkaline single-cell gel electrophoresis). After cell cultures were prepared, test groups were treated with different concentrations of PCB 52 (0.2 and 1 μM) and PCB 77 (1 and 10 μM) for 1 h at 37 °C in a humidified carbon dioxide incubator, and compared to a DMSO vehicle control group. The cells were visually classified into four categories on the basis of extent of migration such as undamaged (UD), low damage (LD), moderate damage (MD) and high damage (HD). The highest concentration of PCBs 52 and 77 significantly increased DNA breakage in human lymphocytes (p < 0.001). Our results indicate that both the non-coplanar PCB 52 and coplanar PCB 77 cause DNA damage, and that the ortho-substituted congener was significantly more potent than the dioxin-like coplanar congener.     

UV-susceptibility of zoospores of the brown macroalga Laminaria digitata from Spitsbergen

The UV-susceptibility of zoospores of the lower sublittoral kelp Laminaria digitata was studied in the laboratory under varying fluence of spectral irradiance consisting of photosynthetically active radiation (PAR, 400–700 nm; = P), PAR + UV-A radiation (UV-A, 320–400 nm; = PA), and PAR + UV-A + UV-B radiation (UV-B, 280–320 nm; = PAB). In vivo absorption of phlorotannin, localisation of phlorotannin-containing physodes, structural changes, DNA damage and repair, photosynthesis and germination of zoospores were measured after exposure treatments and after 2–6 days of recovery in dim white light. Photodegradation of phlorotannins was observed after extended exposure to ultraviolet radiation (UVR). The UV-protective function of extra- and intracellular phlorotannins was, therefore, observed only after 8 h, but not after 16-h UVR exposure. The energetic cost of photoprotection may have caused the delay in ontogenic development of zoospores after 8-h exposure to PA and PAB treatment; longer exposure time corresponding to 16-h PA and PAB treatment eventually lead to cell degeneration at 6 days post-cultivation. The formation of cyclobutane–pyrimidine dimers (CPDs), as indicator of DNA damage, was not blocked by the UV-absorbing phlorotannins during the 16-h PAB exposure and the inability for DNA damage repair was likely responsible for low photosynthetic recovery and spore mortality. The higher sensitivity of L. digitata zoospores to UVR compared to other kelps such as Saccorhiza dermatodea and Alaria esculenta confirmed our hypothesis that the depth distribution of adult sporophytes in the field correlates to the sensitivity of their corresponding early life history stages to different stress factors in general and UVR in particular.     

Protective action of melatonin against oxidative DNA damage—Chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 μM and its direct metabolite N¹-acetyl-N²-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 μM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.     

Main features of the oxidative metabolism in gills and liver of Odontesthes nigricans Richardson (Pisces, Atherinopsidae)

The aim of this work was to study comparatively the oxidative metabolism in gills and liver of a silverside, Odontesthes nigricans, in their natural environment, the Beagle Channel. Oxidative damage to lipids was evaluated by assessing TBARS and lipid radical content, in gills and liver. Gills showed a significantly higher degree of damage than liver. The content of α-tocopherol, β-carotene and catalase activity showed significantly higher values in the liver than in the gills. The ascorbyl radical (A^•) content showed no significant differences between gills and liver. The ascorbate (AH⁻) content was 12 ± 2 and 159 ± 28 nmol/mg FW in gills and liver, respectively. Oxidative metabolism at the hydrophilic level was assessed as the ratio A^•/AH⁻. The ratio A^•/AH⁻ was significantly different between organs, (6 ± 2)10^− 5 and (5 ± 2)10^− 6, for the gills and the liver, respectively. Both, lipid radical content/α-tocopherol content and lipid radical content/β-carotene content ratios were significantly higher in gills as compared to the values recorded for the liver, suggesting an increased situation of oxidative stress condition in the lipid phase of the gills. Taken as a whole, the O. nigricans liver exhibited a better control of oxidative damage than the gills, allowing minimization of intracellular damage when exposed to environmental stressing conditions.     

Impact of maternal lifestyle factors on newborn HPRT mutant frequencies and molecular spectrum — Initial results from the Prenatal Exposures and Preeclampsia Prevention (PEPP) Study

Epidemiological studies have demonstrated associations between maternal tobacco smoke exposure and consumption of alcohol during pregnancy and increased risk of pediatric malignancies, particularly infant leukemias. Molecular evidence also suggests that somatic mutational events occurring during fetal hematopoiesis in utero can contribute to this process. As part of an ongoing multi-endpoint biomarker study of 2000 mothers and newborns, the HPRT T-lymphocyte cloning assay was used to determine mutant frequencies (M_f) in umbilical cord blood samples from an initial group of 60 neonates born to a sociodemographically diverse cohort of mothers characterized with respect to age, ethnicity, socioeconomic status, and cigarette smoke and alcohol exposure. Non-zero M_f (N=47) ranged from 0.19 to 5.62×10⁻⁶, median 0.70×10⁻⁶, mean±SD 0.98±0.95×10⁻⁶. No significant difference in M_f was observed between female and male newborns. Multivariable Poisson regression analysis revealed that increased HPRT M_f were significantly associated with maternal consumption of alcohol at the beginning [Relative Rate (RR)=1.84, 95% CI=0.99–3.40, P=0.052) and during pregnancy (RR=2.99, 95% CI=1.14–7.84, P=0.026). No independent effect of self-reported active maternal cigarette smoking, either at the beginning or throughout pregnancy, nor maternal passive exposure to cigarette smoke was observed. Although based on limited initial data, this is the first report of a positive association between maternal alcohol consumption during pregnancy and HPRT M_f in human newborns. In addition, the spectrum of mutations at the HPRT locus was determined in 33 mutant clones derived from 19 newborns of mothers with no self-reported exposure to tobacco smoke and 14 newborns of mothers exposed passively or actively to cigarette smoke. In the unexposed group, alterations leading to specific exon 2–3 deletions, presumably as a result of illegitimate V(D)J recombinase activity, were found in five of the 19 mutants (26.3%); in the passively exposed group, two exon 2–3 deletions were present among the seven mutants (28.6%); and in the actively exposed group, six of the seven mutants (85.7%) were exon 2–3 deletions. Although no overall increase in HPRT M_f was observed and the number of mutant clones examined was small, these initial results point to an increase in V(D)J recombinase-associated HPRT gene exon 2–3 deletions in cord blood T-lymphocytes in newborns of actively smoking mothers relative to unexposed mothers (P=0.011). Together, these results add to growing molecular evidence that in utero exposures to genotoxicants result in detectable transplacental mutagenic effects in human newborns.     

Combined effects of the UGT1A1 and OATP2 gene polymorphisms as major risk factor for unconjugated hyperbilirubinemia in Indian neonates

Genetic association studies have linked a number of single nucleotide polymorphisms (SNPs) with unconjugated hyperbilirubinemia. The present study was undertaken to validate the association of SNPs with development of hyperbilirubinemia in Indian neonates. Genotyping of five SNPs in two candidate genes was performed in 126 infants with hyperbilirubinemia and 181 controls by PCR-RFLP, Gene Scan analysis and direct DNA sequencing. Genetic polymorphisms of the UGT1A1 promoter, specifically the − 3279 T ? G phenobarbital responsive enhancer module (rs4124874) and (TA)7 dinucleotide repeat (rs8175347) as well as the coding region variants (rs2306283 and rs4149056) of the OATP2 gene were significantly higher among the cases than the controls. The presence of the mutant haplotypes either in homozygous, heterozygous or compound heterozygous state had a significant effect on neonatal hyperbilirubinemia as well as on the requirement of phototherapy than those with the wild haplotype. Further, a significantly higher number of hyperbilirubinemic cases had ≥ 3 variants than the controls (73.80% vs 40.36%, p < 0.0001) and the mean total serum bilirubin levels and requirement of phototherapy also increased according to the number of variants co-expressed. This study demonstrates that UGT1A1 and OATP2 polymorphisms were associated with altered bilirubin metabolism and could be genetic risk factors for neonatal hyperbilirubinemia.     

A proteomic approach to the bilirubin‐induced toxicity in neuronal cells reveals a protective function of DJ‐1 protein

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long‐term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB‐induced neuronal toxicity, we used the human neuroblastoma cell line SH‐SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ‐1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin‐induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.     

Effect of phototherapy on oxidant/antioxidant status: a randomized controlled trial

In order to evaluate the effect of different types of phototherapy on oxidant/antioxidant status in hyperbilirubinemic neonates, an interventional randomized control trial was conducted on 120 neonates ≥35 weeks’ gestational age with indirect hyperbilirubinemia reaching phototherapy level. This study is registered with ClinicalTrials.gov as NCT03074292. Neonates were assigned to three groups; 40 neonates received conventional phototherapy, 40 received intensive phototherapy and 40 received blue light-emitting diodes (LED) phototherapy. Complete blood count (CBC), total serum bilirubin (TSB), total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO), copper (Cu), zinc (Zn), and iron (Fe) levels were measured before and 24?hours after phototherapy. TSB decreased postphototherapy in all three groups (p < .05 for all), with significantly lower levels following intensive and LED phototherapy compared to conventional phototherapy (p < .05 for both). TAC decreased postphototherapy in the three groups (p < .05 for all). MDA and NO increased postphototherapy (p < .05 for all), with the intensive phototherapy group having the highest levels followed by the conventional while LED phototherapy group showed the lowest levels in comparison to the other groups (p < .05). Cu, Zn and Fe increased postphototherapy in all three groups (p < .05 for all). Positive correlations were found between postphototherapy TSB with TAC, Cu and Zn (p < .05) and negative correlations with MDA, NO and Fe (p < .05) among neonates of the 3 studied groups. In conclusion, different photo therapies have an impact on oxidant/antioxidant balance and are associated with increased oxidative stress markers with the LED phototherapy being the safest.     

Optimizing Exchange Transfusion for Severe Unconjugated Hyperbilirubinemia: Studies in the Gunn Rat

Background

Severe unconjugated hyperbilirubinemia carries the risk of neurotoxicity. Phototherapy (PT) and exchange transfusion (ET) are cornerstones in the treatment of unconjugated hyperbilirubinemia. Studies to improve ET efficacy have been hampered by the low application of ET in humans and by the lack of an in vivo model. The absence of an appropriate animal model has also prevented to determine the efficacy of adjunct or alternative treatment options such as albumin (Alb) administration.

Aim

To establish an in vivo model for ET and to determine the most effective treatment (combination) of ET, PT and Alb administration.

Methods

Gunn rats received either PT, PT+Alb, ET, ET+PT, ET+PT+Alb or sham operation (each n = 7). ET was performed via the right jugular vein in ∼20 min. PT (18 µW/cm²/nm) was started after ET or at T₀. Albumin i.p. injections (2.5 g/kg) were given after ET or before starting PT. Plasma unconjugated bilirubin (UCB), plasma free bilirubin (Bf), and brain bilirubin concentrations were determined.

Results

We performed ET in 21 Gunn rats with 100% survival. At T₁, ET was profoundly more effective in decreasing both UCB −44%, p<0.01) and Bf −81%, p<0.05) than either PT or PT+Alb. After 48 h, the combination of ET+PT+Alb showed the strongest hypobilirubinemic effect (−54% compared to ET).

Conclusions

We optimized ET for severe unconjugated hyperbilirubinemia in the Gunn rat model. Our data indicate that ET is the most effective treatment option, in the acute as well as the follow-up situation.