JACK D. THRASHER, Ph.D

(505) 937-2250 - Cell

 Email: toxicologist@drthrasher.org

    Somatic mutations have been demonstrated in mtDNA from bleomycin,⁵ Psoralen,⁶ oxidants,⁷ aging and degenerative diseases.^8-10 sunlight exposure,¹¹ cigarette smoking,¹² oxygen and oxidative stress,^13,14 lipid peroxidation¹⁵, azidothymidine (AZT),¹⁶ antibiotics,¹⁷ and methanol/cyanide exposure.¹⁸

    MtDNA mutations are responsible for several mitochondrial syndromes that are inherited maternally.^{3,  4}

    However, search of the literature has not revealed a cause for maternal mtDNA mutations.  In this editorial, I will describe 5 cases of global developmental delay following exposure of mothers to several organochlorine (OCPs).  I hope the information herein will stimulate other researchers to investigate the effects of exposure to xenobiotics on maternal mtDNA and birth defects.

    Five children (3 girls – aged 2 y, 3 y and 5 y; and 2 boys – aged 2 y and 5 y) were diagnosed with severe global developmental delay and hypotonia.  One girl and 1 boy, had deficiencies in complexes I and III, and I, respectively.  One boy had carinitine deficiency with early myopathy and a suspected mitochondrial defect; currently he awaits mitochondrial test results.  The other 2 girls were severely developmentally delayed, and 1 had an abnormality in beta-hydroxybutyrate and related metabolites.  The treating physicians suspected mitochondrial defects.

    The 4 mothers lived in the contaminated area as follows: (a) 2 sisters (30 y and 32 y of age), respectively lived in the contaminated area for 10 y (i.e. ages 2 y – 12 y and 4 y – 14 y, respectively): (b) 1 woman resided in the area from age 14 y until age 19 y; and (c) 1 woman who moved to the area at age 6 y or 7 y continues to reside in the area.  There is no family history of birth defects in any of the women or their husbands.  The husbands were not born or raised in the contaminated area.  Furthermore, the earlier-referenced sisters (i.e., item [a]) had 2 half-sisters who were 14 y and 16 y older than they, and the half-sisters were not raised in the contaminated area; the 5 children of the half-sisters did not have global developmental delay.   No fat biopsies or blood level measurements were performed for either mothers or children.

    1. mtDNA mutations have been observed in human oocytes and embryos.  Twenty-three novel mtDNA rearrangements – with deletions, insertions, and duplications – were found and were reportedly unrelated to aging.  Significant reductions in the number of oocytes containing mtDNA rearrangements occurred as the oocyte developed from the germinal vesicle to the mature metaphase oocyte.^{20, 21}

    2.  Investigators have reported that OCPs occur in human follicular fluid in the ranges of parts per trillion to (ppt) to parts per billion (ppb).^{22,  23}  Foster et al²³ blamed the failure of in vitro fertilization on OCPs in follicular fluid.

    3.  The ovaries of Rhesus monkeys exposed to HCB resembled human menopausal ovaries.  Degenerative changes were observed in the germinal epithelium and ovarian stroma, and a reduction in primary follicles was also noted.  Tissues of baby monkeys, which were breast-fed by exposed mothers, contained 2.5-5.5 times more HCB than maternal tissues.  HCB caused toxic effects in primordial germ cells at concentrations that did not cause toxic hepatic effects.^24-27

    4.  Cynmolagous monkeys, exposed to 0.01 mg HCB/kg body weight (bw), 0.1 mg HCB/kg bw, and 10.0 mg HCB/kg /bw, had histopathologic and ultrastructural changes in their ovaries.  Degenerative changes in the thecal and germinal epithelium occurred at all concentrations.  Ultrastructural abnormalities were observed in mitochondria of ova and follicular cells.  The changes in ovarian structures and ova mitochondria occurred at doses that ranged from ppt to ppb.^{28, 29}

    5.  OCPs have been found in human placenta³⁰ and in human umbilical cord bood in mother/child pairings.³¹

    6.  OCPs have been isolated from human milk that was reconstituted to concentrations found in the milk and fed to newborn mice.  This resulted in reduced white blood cell counts, as well as in toxic effects to liver smooth endoplasmic reticulum and mitochondria.  These effects were observed at doses in the ppb to ppm range.³²

    7.  OCPs uncouple oxidative phosphorylation, cause oxidative stress, bind to protein complexes and submitochondrial fractions, and alter mitochondrial morphology and function.^33-42

    8.  Mutations have been induced in primordial male and female germ cells,⁴³  oocytes,⁴⁴  and zygotes^45-50 of mice.  Mice treated with ethylnitrosurea produced male offspring with protein variants associated with microsomal and mitochondrial fractions.⁵¹   These variants were transmitted with either a Mendelian or non-Mendelian inheritable pattern.  It should be noted that some zygotic mutations are not typically the result of usual genetic causes.⁵⁰

    9.  In mouse zygotes, the cytoplasm (mitochondria) mediates both development and oxidation-induced apoptic cell death.⁵²   Furthermore, several different chlorinated pesticides cause generation of reactive oxygen species, DNA damage, and lactate dehydrogenase leakage.  These reactive species may serve as common mediators of apoptosis (programmed cell death).⁵³

    In conclusion, the observations on these 5 children and their mothers revealed some interesting facts.  First, the children had similar symptoms that occur with global developmental delay and hyoptonia.  Two of the children were diagnosed with mitochondrial defects (complex I, and I and III, whereas 2 others have suspected mitochondrial defects (i.e. carnitine deficiency and abnormalities of beta-hydroxybutyrate).  The 5^th child, a 2 y old female, required additional testing.  Second, mothers spent some of their childhoods in the contaminated area.  Third, there were no paternal or maternal family histories of developmental defects.  Fourth, the 4 fathers neither were raised nor lived near the contaminated site.  These facts suggest that the mitochondrial defects and the global developmental delay were environmentally related and were inherited maternally.

    Supporting evidence for the above suggestions originates from scientific and medical literature and includes the following: (a) there is a presence of OCPs in human placenta and umbilical cord blood, as well as in follicular fluid; (b) OCPs produce adverse effects on the ovaries and primordial germ cells of old-world monkeys (old-world monkeys have a menstrual cycle similar to humans); (c) OCPs have an effect on mitochondrial structure and function; (d) OCPs effects neonatal mice; (e) some OCPs are known mutagens; (and (f) mtDNA mutations occur in human oocytes and are unrelated to aging.  These points are particularly important because mitochondria, under oxidative stress, produce free radicals.  Free radicals are suspected mutagens and may cause mtDNA mutations that alter mitochondrial function.  The somatic mtDNA mutations listed above are apparently related to the production of free radicals and oxidative stress.

    Investigators must conduct further research into the causation of maternal mtDNA mutations – particularly following exposure to mutagenic xenobiotics – and into assessment of the roles of placental transfer of OCPs, as well as determine the effect of breast-feeding on mitochondrion function and human development.  We know that it is more efficient to identify inducers of disease and to initiate preventative measures than to seek cures.

    1. Anderson S, Bankier J, Arrell CG, et al.  Sequence and organization of the human mitochondrial genome.  Nature 1981; 290:457-65.

   2.  Wallace DC.  Report of the committee on human mitochondrial DNA.  Cytogenet Cell Genes 1990; 55:396-405.

   3.  Wallace DC.  Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Sci 1994; 91:8739-46.

   4.  Giles RE, Blanc H, Cann M, et al. Maternal inheritance of human mitochondrial dNA.  Proc Natl Acad Sci 1980; 77:67815-19.

   5.  Shen C-C, Wertelecki W, Driggers WJ, et al.  Repair of mitochondrial DNA damage by Bleomycin in human cells. Mutat Res 1995; 337:19-23.

    6.  Cullinane C, Bohr VA.  DNA intersrand cross-links induced by psoralen are not repaired in mammalian mitochondria.  Cancer Res 1998; 48:1400-04.

   7.  Ritcher C, Gogvadze V, Laffranchi R, et al.  Oxidants in mitochondria: from physiology to diseases.  Biochim Biophys Acta 19995; 1271:74-76. 

   8.  Ozawa T.  Mechanism of mitochondrial DNA mutations associated with age and diseases.  Biochim Biophys Acta 1995 1271:177-89.

   9.  Wallace DC, Shoffner JM, Trounce IT, et al.  Mitochondrial DNA mutations in human degenerative diseases and aging.  Biochim Biophys Acta 1995; 1271:141-51

    10.  Corral-Debrinski M, Shoffner JM, Lott MT, et al.  Association of mitochondrial DNA damage with aging and coronary atherosclerotic heart disease.  Mutat Res 1992; 275:169-89.

    11.  Pang C-Y, Lee H-C, Yang J-H, et al.  Human skin mitochondrial DNA deletions associated with light exposure.  Arch Biochem Biophys 1994; 312:534-38.

    12.  Ballinger SC, Coude TG, Davis GS, et al.  Mitochondrial genome damage associated with cigarette smoking.   Cancer Res 1996; 56:92-97.

    13.  Yoneda M, Kaatsumata K, Hayakawa M, et al.  Oxygen stress induces an apoptotic cell death associated with fragmentation of mitochondrial genome.  Biochem Biophsy Res Comm 199; 209:723-29.

    14.  Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.  Proc Natl Acad Scie 1997; 94:514-19.

    15.  Hruszkewycz AM.  Lipid peroxidation and mtDNA degeneration: a hypothesis.  Mut Res 1992; 275:243-48. 

    16.  Dalakas MC, Leon-Monzon ME, Bernardini I, et al.  Zidovudine-induced mitochondrial myopathy is associated with muscle carnitine deficiency and lipid storage.  Ann Neurol 1994; 25:482-87.

    17.  Prezant TR, Agapian IV, Bhohlman MC, et al.  Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness.  Nat Genet 1993; 4:289-94.

    18.  Sadun A.  Acquired mitochondrial impairment as a cause of optic nerve disease.  Trans Am Opthalmol Soc. 1998; 96:881-923.

    19.  Merchant R.  Public Health  Assessment.  Landia Chemical Company and Florida Favorite Fertilizer, Lakeland, Polk County, Florida, CERCLIS NO. FL042110841.  Lakeland, FL:Bureau of Environmental Toxicology, Florida Department of Health.

    20.  Brenner CA, Wolny YM, Barritt JA, et al.  Mitochondrial DNA deletion in human oocytes and embryos.  Mol Human Repro.  1998; 4:887-92.

    21.  Barritt JA, Brenner CA, Cohen I, et al.  Mitochondrial DNA rearrangements in human oocytes and embryos.  Mol Human Repro 1999; 5:927-33.

    22.  Trapp M, Bakoloh V, Bohnet H-G, et al.  Pollutants in human follicular fluid.  Fertil Steril 1984; 42:146-48.

    23.  Foster WG,  Jarrell  JF, Younglai EV, et al.  An overview of some reproductive toxicology studies conducted at Health Canada.  Toxicol Ind Health 1996; 12:447-59.

    24. Iatropoulous MJ, Hobson W, Knauf V, et al. Morphological effects of hexachlorobenzene toxicity in female Rhesus monkeys.  Toxicol Appl Pharmacol 1976; 37:433-44.

    25.  Muller WF, Hobson W, Fuller GB, et al.  Endocrine effects of chlorinated hydrocarbons in Rhesus monkeys.  Ecotox Environ Safety. 1978; 2:161-72.

    26.  Muller WF.  Fate and effects of hexachlorobenzene in non-human primates and other laboratory animals.  International Agency for Cancer Research (IARC Monograph No. 77. Lyon, France:IARC, pp 287-88.

    27.  Jarrell JF, McMahon A, Villeneuve D, et al.  Hexachlorobenzene toxicity in monkey primordial germ cells without induced porphyria.  Repro Toxicol 1993; 7:41-47.

    28.   Babineau A, Singh A, Jarrell IF, et al. Surface epithelium of the ovary following oral administration of hexachlorobenzene to the monkey.  J Submicro Cytol Pathol 1991; 23:457-64.

    29.  Bourque AC, Singh A, Lakhanpal N, et al.  Ultrastructural changes in ovarian follicles of monkeys administered hexachlorobenzene.  Am J Vet Res. 1995; 56:1673-77.

    30.  McLeod HA, Grant DL, Phillips WEJ.  Pesticide residues and metabolites in placentas. Caan J Public Hlth 1971; 62:3441-44.

    31.  Saxena MC, Siddiqui MKJ, Bhargava AL, et al.  Placental transfer of pesticides in humans.  Arch Toxicol. 1981; 48:127-34.

    32.  Lembowicz K, Sitarskaa E, Gorski T, et al.  The effect of organic chlorine compounds and their metabolites present in human milk on newborn mice.  Toxicol Lett 1991; 57:215-26.

    33.  Trenti T, Ventura E, Ceccarelli D et al. Functional derangement of liver mitochondria from hexachlorobenzene-treated rats.  Internal Agency For Research on Cancer )IARC Monograph No. 77.  Lyon, France:IARC, pp 329-31.

    34.  Sahoo A, Chainy GBN.  Acute hexachlorobenzene-induced oxidative stress in rat cerebral hemisphere.  Neurochem Res 1998; 23:1079-84.

    35.  Bczkowski JZ.  The mode of action of p,p’DDT on mammalian mitochondria.  Toxicology 1976; 6:309-14.

    36.  Cherfurka W, Gnidec EP.  Binding of [14C] DDT by submitochondrial particles.  Comp Biochem Physiol C 1987; 88:213-73.

    37.  Moreno AJM, Madeira MC.  Mitochondrial bioenergetics affected by DDT.  Biochim Biophys Acta 1991: 1060:166-73.

    38.  Ferriera FM. Maddeira VM, Moreno AL.  Interaction of 2,2bis(p-chlorophenyl)-1,1-dichloroethylene with mitochondrial oxidative phosphorylation.  Biochem Pharmacol 1997; 53:299-308.

    39.  Martz F, Straw JA.  Metabolism and covalent binding of 1-(o-chlorophenyl)-1-)p-chlorophenyl0-2,2-dichloroethane (o,p’-DDD).  Correlation between adrenocorticolytic activity and metabolic activation of adrenocortical mitochondria. Drug Metab Dispos 1980; 8:127-30.

    40.  Junqueira VB, Koch OR, Arisi AC, et al.  Regression of morphological alterations and oxidative stress-related parameters after acute lindane-induced hepatotoxicity in rats.  Toxicology 1997; 117:199-205.

    41.  Mhrotra BD, Bansal SK, Desaiah D. Compative effects of structurally related cyclodiene pesticides on ATPases.  J Appl Toxicol 1982; 2:278-83.

    42.  Trottman CH, Prasada Rao KS, Morrow W, et al.  In vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues.  Life Sci 1985; 36:427-33.

    43.  Shibuya T, Murota T, Horiya N, et al.  The induction of recessive mutations in mouse primordial germ cells with N-ethy-N-nitrosurea.  Mutat Res 1993; 290:273-80.

    44.  Russell,LB, Russell WL.  Frequency and nature of specific-locus mutations induced in female mice by radiations and chemicals: A review.  Mutat Res 1992; 296:107-27.

    45.  Russell LB, Bangham JW, Stertzner KF, et al.  High frequency of mosaic mutants produced by N-ethyl-N-nitrosurea exposure of mouse zygotes.  Proc Natl Acad Sci 1988; 85:167-70.

    46.  Russell LB, Bangham JW.  The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome. Mutat Res 1991; 248:203-09.

    47.  Generoso WM, Shourbaji AG, Piegorsch WW, et al.  Developmental response of zygotes exposed to similar mutagens.  Mutat Res 1991; 250:439-46.

    48.  Generoso WM, Rutledge JC, Cain KT, et al.  Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death. Mutat Res 1987; 176:269-74.

    49.  Generoso WM, Rutledge JC, Cain KT, et al.  Mutagen-induced fetal anomalies and death following treatment of females within hours after mating.  Mutat Res 1988; 199:175-81.

    50.  Katoh M, Caheiro NLA, Cornett CC, et al.  Fetal anomalies produced subsequent to treatment of zygotes with ethylene oxide or ethyl methanesulfaonate are not likely due to the usual genetic causes.  Mutat Res 1989; 210:337-44.

    51.  Giometti CS, Gemmell MA, Tollaksen SL, et al. Heritable protein variants induced by exposure to ethylnitrosurea: heritability, subcellular location, and tissue distribution.  Mutat Res 1988; 2202:9-17.

    52. Lin L, Keefe DI.  Cytoplasm mediates both development and oxidation-induced apoptic cell death in mouse zygotes. Biol Repro 2000; 62:1828-34.

    53.  Bagchi D, Bagchi M, Hassoun EA, et al.  In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides.  Toxicology  1995; 104:128-40.