Embryo Toxicity and Teratogenecity of Formaldehyde (FA)

Jack D. Thrasher, Ph.D.
Sam-1 Trust
Alto, New Mexico 88312

Kaye H. Kilburn, M.D.

University of Southern California
Keck School of Medicine
Environmental Sciences Laboratory
2025 Zonal Avenue, CSC 201

Los Angeles, California 90033

ABSTRACT

C-14 FA crosses the placenta and enters fetal tissues. The incorporated radioactivity is higher in fetal organs (brain and liver) than in the maternal tissues. The incorporation mechanism has not been fully studied, but FA enters the single carbon cycle, and is incorporated as a methyl group into nucleic acids and proteins. Also, FA chemically reacts with organic compounds (DNA, nucleosides, nucleotides, proteins, amino acids) by addition and condensation reactions, forming adducts and DNA-protein crosslinks. The following questions need answered: What adducts (e.g. N-methyl amino acids) are formed in the blood following FA inhalation? What role do N-methyl-amino adducts play in alkylation of nuclear and mitochondrial DNA as well as mitochondrial peroxidation? The fact that the free FA pool in the blood is not affected following exposure to the chemical, does not mean that FA is not involved in altering cell and DNA characteristics beyond the nasal cavity.

Teratogenic effect of FA in the English literature has been sought beginning the 6th day of pregnancy (rodents).79-82 The exposure regimen appears to be critical and may accout for the differences in outcomes. Pregnants rats were (1) either exposed prior to mating, mated, and during the entire gestation period or (2) were exposed during the entire gestation period. These regimens increased embryo mortality, fetal anomalies (cryptochordism, aberrant ossification centers), decreased concentrations of ascorbic acid, and abnormalities in enzymes of mitochondria, lysosomes and ER. The alterations in enzymatic activity persisted four months after birth. In addition, FA caused a metabolic acidosis, which was augmented by iron deficiency. Furthermore, newborns exposed to FA in utero had abnormal performances in open-field tests.

Disparities in teratogenic effects of toxic chemicals are not unusual. For example, Chlorpyrifos has been shown to not have teratogenic effects in rats when mothers are exposed on days 6 through 15(16) of gestation.83,84 However, either changing the end-points for measurement or exposing neonates during periods of neurogenesis (days 1-14 after birth) and later developmental periods, produced adverse effects. These included neuro-apoptosis, decreased DNA and RNA synthesis, abnormalities in adenylyl cyclase cascade, and neurobehavioral effects.85-92 Furthermore, the terata caused by thalidomide is a graphic human example in which the animal model and timing of exposure were key factors.93,94 Thus, it appears that more sensitive end-points (e.g. enzyme activity, generation of reactive oxygen species, timing of exposure, etc.) for the measurement of toxic effects of environmental agents on embryos, fetuses and neonates are more coherent than are gross terata observations.

The perinatal period from the end of organogenesis to the end of the neonatal period in humans approximates the 28th day of gestation to 4 weeks post partum. Therefore, it is of primary concern to investigate similar stages of development, e.g. neurogenesis occurs in the 3rd trimester in humans and neonatal days 1-14 in rats and mice, while guinea pigs are more like humans.

Finally, screening for teratogenic events should also include exposure of females prior to mating or shortly after mating. Such a regimen is fruitful since environmental agents do cause adverse effects on ovarian elements, e.g. thecal cells and ova (nDNA and mitDNA) as well as zygotes and embryos prior to implantation. mtDNA mutations and deletions occur in human oocytes and embryos .93,94 Thus, it is probable that xenobiotics directly effect nDNA and/or mitDNA in either or both the ovum and zygote/embryo 96 and could account for the increasing appearance of a variety mitochondrial diseases, including autism.97-99

Two cases of human birth defects were reported in FA-contaminated homes.100 One was anencephalic at 2.76 ppm, and the other defect at 0.54 ppm was not characterized. Further observations on human birth defects are recommended.

Introduction

Formaldehyde (FA) is widely spread in the environment, used to manufacture wide variety of products, and its major uses are ureaformaldehyde (UF) resins (25%), phenol-FA (PhF) resins (20%), plastics (15%) and intermediates (22%). It is an intermediate in acetylinic chemicals, and used to produce of 4,4′-hexamethlenetetramine, UF concentrates, 4,4′-methylenediphenyl diisocyanate, chelating agents, and trimethylolpropane. UF and PhF resins are used primarily as adhesives in the manufacture of particle board, fiberboard, plywood, molding, paper treating and coating, textile treating, surface coating and fiberglass insulation.1 OSHA estimates that approximately 2.1 million workers are exposed to formaldehyde (1,2). Domestic exposures occur mainly from consumer products that include textiles (clothing and household furnishings), insulation (fibrous and foams), paper, cosmetics and wood-products (particle board, plywood, medium density fiber board) (3).

The current OSHA permissible exposure limit (PEL) is 0.75 ppm as an 8-hour time weighed average (TWA). This standard includes a 2 ppm short-term 15 minute exposure limit (STEL) with an ‘action level’ of 0.5 ppm measure over 8 hours.2 Other exposure levels are: ACGIH TLV of 0.3 ppm (0.37 mg/m3 ceiling); NIOSH REL of 0.016 ppm (TWA) and 0.1 ppm (15 minute ceiling). FA is classified as a probable human carcinogen: USEPA (Class 2A); IARC (Class 2A), OSHA (Carcinogen); NIOSH (Carcinogen) and NTP (Reasonably anticipated as a carcinogen) 4.

Acute health effects and doses are: odor threshold (0.05-1.0 ppm); eye irritation (0.01-2.0 ppm); irritation of eyes/nose/throat/upper respiratory system (1.0-3.0 ppm); intolerable; (4.0-5.0 ppm); severe respiratory symptoms/difficulty breathing (10-20 ppm); serious respiratory tract injury (>50 ppm); and death (>100 ppm). The IDLH is 20 ppm for an exposure of 13-30 min. Chronic exposure to FA can lead to dermal and respiratory sensitization, lower airway and chronic pulmonary obstruction and immunologic manifestations.5,6,7 Evaporation from formalin at 20 oC yields 5 ppm of FA.

Reproductive and developmental effects are believed to be minimal. This conception is based on a few epidemiological studies and the absent of birth defects in animal studies following FA exposure,8 as a result of reviews by EPA9 and WHO10 of the scientific/medial literature prior to 1989. More recent epidemiologic investigations have shown exposure to FA is associated with delayed conception,11 and an increased risk of spontaneous abortion in wood workers,11 laboratory personnel 12 and cosmetologist.13 Reports from Japanese and Russian literature on the embryotoxicity of FA in rodents demonstrate that FA crosses the placenta to the fetus, causes birth defects and affects on enzyme function in mitochondria, lysosomes and endoplasmic reticulum. This communication reviews this research with critizques in the perspective of the current scientific knowledge of the biological chemistry of FA.

Distribution of 14CFA in Maternal and Fetal Tissues

The uptake and distribution of C14-labeled FA (14CFA) was studied in pregnant ICR mice.14-16 On the sixteenth day of gestation adult pregnant mice were injected via tail vein with 0.05 ml of 1 % formalin containing 3.5 mg of 14CFA. The animals were killed at intervals from 5 minutes up to 48 hours. The incorporation of 14CFA and its metabolites was followed by frozen section autoradiography and liquid scintillation detection. Control measure was taken to avoid errors due to loss of radioactivity by volatility of the 14CFA and its metabolites from frozen sections. TCA treated maternal and fetal liver showed the incorporation of the isotope into DNA and acid insoluble fractions.

The autoradiograms and scintillation counting demonstrated a rapid uptake (by 5 minutes) of 14CFA into maternal liver, lung, heart, salivary gland, gall bladder, spleen kidney, bone marrow, nasal mucosa, uterus, placenta and fetal tissues (Table 1). The radioactivity appeared in the urine and feces up to 6 hours after injection.

Incorporation of the labeled isotope was greater in the placenta, uterus and fetal tissues than in other maternal organs (Table1). The fetal brain had significantly greater uptake than the maternal brain at six hours after injection. At 48 hours residual radioactivity in mother and fetus was 29.6 % of the administered dose. The remaining 14CFA was either expired or excreted in urine and feces. The DNA fraction contained 20 % and 50 % of the total radioactivity in maternal and fetal liver at 6 and 24 hours after injection. (Fig.1).

Elimination of FA and its metabolites from fetal tissues was slower than maternal tissues. This was particularly evident in fetal liver and brain. Moreover, the radioactivity in the fetal brain was twice that observed in the maternal brain at 6 hours and afterward (Table 1).

Table 1. Maternal and fetal concentrations of radioactivity in pregnant mice
Time

After

Injection

Tissue

Source

Blood

Brain

(whole)

Liver

Placenta

Fetus

(whole)

Amnion

(fluid)

Amnion

Uterus
5 min

Maternal

Fetal

154.4+39.9

—–

36.7+4.6

31.8+10.4

307.9+58.8

88.9+39.3

65.5+27.4

—–

—-

63.1+30.6

39.2+33.8

—-

56.8+23.8

—-

141.9+37.3

—-
30 min

Maternal

Fetal

79.0+13.4

—-

48.7+3.1

47.7+20.1

269.3+43.3

129.0+42.5

75.3+22.8

—-

—-

81.1+19.7

19.3+6.9

—-

53.9+9.9

—-

110.2+29.9

—-
1 hr

Maternal

Fetal

117.1+24.1

—-

44.6+0.5

56.2+34.6

298.2+66.7

150.3+4.3

89.9+19.7

—-

—-

85.1+32.0

14.6+7.9

—-

58.4+29.1

—-

109.3+26.5

—-
3 hr

Maternal

Fetal

149.4+18.9a

—-

53.1+25.6

71.0+24.2

361.8+64.5

263.9+56.7

120.1+23.8

—-

—-

117.7+26.5

18.8+11.3

—-

80.2+21.1

—-

135.5+27.8

—-
6 hr

Maternal

Fetal

48.8+3.8

—-

22.9+6.0

35.0+8.0b

180.4+42

142.5+38.1

62.6+0.11

—-

—-

79.5+25.7

8.9+4.9

—-

51.1+17.0

—-

82.4+10.4
24 hr

Maternal

Fetal

15.4+5.8

—-

17.7+4.2

36.5+12.1c

105.7+37.7

96.2+38.6c

48.5+21.3

—-

—-

65.0+29.0

4.9+1.9

—-

45.3+21.1

—-

69.3+20.1

—-
48 hr

Maternal

Fetal

9.0+2.5

—-

9.5+2.5

22.3+5.9

48.3+10.8

48.2+16.6

25.9+4.8

—-

—-

—-

—-

—-

—-

—-

—-

—-

ap<0.05, significantly different from radioactvity at 30 minutes; bp<.05, cp<.01, significantly different from maternal
radioacitivty. The results are expressed in dpm/mg (ul). Mean+SD. n = 5

Cytopathic and Cytogenetic Effects of FA Inhalation on Germ Cells and Bone Marrow Cells.

Adult female Wistar rats (experimental and control) were housed under controlled lighting (12 hrs light/12 hrs dark) and given free access to food and water.17 Females were exposed to FA by inhalation 4 hrs/day in special chambers for 4 months (except on working days) at 0.5 and 1.5 mg/m3 of air. Exposed and control females were mated with intact males after exposure. Embryos were extracted from uterii by saline solution lavage on days 2 and 3 of pregnancy. The morphology of embryos was observed by MBS-9 and MB1-11 (phase-contrast illumination) microscopy. A portion of the embryos was used for making up total Tartosky preparations.

Bone marrow was taken from the same animals at 48-72 hrs after termination of FA exposure and prepared for cytogenetic studies by the standard method. One hundred metaphases were analyzed per animal from coded preparations for the mitotic index. All types of chromosomal aberrations and cells containing from 40-43 chromosomes were recorded (gaps were not included). In addition, the dependence of the frequency of breaks in chromosomes on their lengths was established. The significance of difference in mean values was assessed using a Student t-test, and differences in proportions with the chi-square test.

No significant effect of 0.5 mg/m3 of FA was observed on embryonic development in the 3rd day of pregnancy. But at 1.5 mg/m3 there was damage to structure (roughness of cytoplasm, pyknosis of nuclei) of blastomeres, and increased proportion of degenerating embryos (p <0.05).

The number of metaphases with chromosome aberrations at 0.5 mg/m3 was significantly greater than controls (p <0.05) and was elevated at the higher concentration (p<0.01) (Table 2). In addition, the number of chromosomes with aberrations and aneuploidy was significantly elevated (p <0.05). The mitotic index was decreased at 0.5 mg/m3 (p <0.05) and increased at 1.5 mg/m3 (p <0.05) when compared to the controls

Table 2. Frequency of chromosomal dammage and aneuploidy in the bone marrow cells of rats that had been subjected to exposure to formaldehyde (FA)
Conc.

of FA

mg/m3

No. of

metaphases

analyzed

No. of

cells with

aberrations

No. aberrations per

100 metaphases

chromatids

No. aberrations per

100 metaphases

chromosomes

No. aneupoid

cells, chromosomes

less than 42

No aneuploid

cells, chromosomes

more than 42

Mitotic

index

(%)
0

600

0.7+0.3

0.3+0.2

0.3+0.2

7.0+1.0

0.2+0.1

5.0+0.3
0.5

785

2.4+0.5a

2.3+0.6

0.2+0.2

10.9+1.1a

0.8+0.3

4.2+0.2a
1.5

625

4.0+0.7b

2.7+0.6a

1.6+0.5a

13.6+1.4a

0+0.04

6.7+0.3a

Note: Differs reliably from control group: ap <0.05; bp<0.01.

The frequency of chromosome breaks was proportional to length. At the same time, the breaks were more frequently in the centromere region (62 %) in metacentric chromosomes and in the telomere region (52 %) in acrocentric chromosomes. An increase in the frequency of genome mutations, i.e. the number of hypoploid cells was also observed in the experimental rats.

FA at low doses possesses cytopathogenic and mutagenic effects and that the cells studied had different levels of susceptibility to the harmful effects of the chemical. The bone marrow cells were more sensitive to the effects of FA than were the germ cells. Bone marrow cells exhibited chromosome aberrations, aneuploidy and changes in the mitotic index. The morphological damage detected in the embryos is not specific to FA, since such degeneration followed exposure to gasoline and contraceptives as previously reported.

It is hypothesized that FA (and possibly many other substances) affect ovarian follicules (differentiation of follicle somatic cells), leading to a disruption egg maturation (final stages of gametogenesis), and impairment of fertilization and early embryonic development.

Prolonged inhalation of FA (near maximum allowable concentrations) affects bone marrow, possibly ovarian elements, zygote and early embryogenesis probably by impairing proliferative activity and chromosome damage (aberrations, aneuploidy).

Effects of FA inhalation on Ascorbic Acid, Nucleic Acids, Organ Weights and Pathology in New Born Rats

The effects of FA inhalation on several fetal parameters haven been reported.18-20 Female adult rats were exposed for 10-15 days by inhalation of formaldehyde at 0.012 and 1.0 mg/m3, then joined with unexposed males, mated, and exposed throughout gestation. Control pregnant females were handled in the same manner for food, temperature, humidity, velocity of air movement, except purified air was supplied to the exposure chamber. The animals were killed at parturition.

Duration of pregnancy was increased by 14-15%, but animals per liter was decreased at 0.012 mg/m3 (9.8) and at 1.0 mg/m3 (8.6) compared to controls (11.3). AA content of the whole fetus, fetal liver and maternal liver was decreased when compared to controls (Table 3). In newborns a statistically significant increase in body weight and weights of thymus, heart, kidney and adrenals, but a decrease in lung and livers were found (Table 3). The authors stated that exposure to FA lowered DNA content and increased RNA content in fetal organs (data not shown). At 1.0 mg/m3 there was an involution of lymphoid tissues, mild hypertrophy of Kupffer’s cells and numerous extra-medullary myelopoietic centers and histochemistry reduced glycogen content of the myocardium and liver, accumulation of positive Schiff’s reaction product in the kidney, and presence of iron in Kupffer’s cells.

Table 3. The effects of exposure to formaldehyde (FA) vapor on weight of organ weights of new born and ascorbic aicd (AA) content in whole fetus, placenta and liver of mothers. The body weight is in grams, organs weights in mg per 10 g body weight, and AA content in mg% (M+sd).
Weights

FA (mg/m3)

(FA mg/m3)

Control
(Organs)

0.012

1.0


Total body

6.0c

6.3b

5.6
Thymus

25.1

31.7c

26.0
Heart

61.5

64.5

61.4
Lung

230.2c

223.2c

287.1
Liver

557.9a

550.8b

587.7
Adrenals

4.2c

3.8a

3.2
Kidney

53.4

55.7b

51.4
Ascorbid Acid

AA (mg/%)

AA(mg/%)

Control
Whole fetus

14.4+0.2c

14.3+0.7b

19.0+1.1
Placenta

6.8+0.7

6.4+0.7

9.7+1.4
Maternal liver

18.1+2.1b

16.8+1.1a

20.6+1.7
Fetal liver

20.1+1.7a

14.8+0.7

15.8+0.4

Note: a = p at 0.05; b = p at 0.01, c = p at 0.001

Embryotoxic Effects of FA on Marker Enzymes of Intracellular Organelles.

Pregnant rats received an aqueous solution of 8 mg/kg (1/50 of LD 50) of FA intragastrically (once/day) throughout pregnancy until killed on the 20th day of gestation (21). The indices of overall embryo mortality and pre- and post-implanation mortality were calculated and anatomical defects recorded. Activity of several enzymes and N-acetyl-neuraminic aicd concentrations were done in fetal and liver tissues (Table 4).

FA caused an increase in pre- and post-implantation mortality as well as increase in embryo mortality by a factor of 2. Mitochondrial enzymes, Malate dehydrogenase (MDH) by 30 % (p = <0.05), and succinate dehydrogenase (SDH) by 50 % (p = <0.05), had decreased activity, while glutamate dehydrogenase increased. FA affected mitochondrial inner membrane permeability, oxidative phorphorylation and impairment of energy production.

Microsomal and lysosomal Inosine diphosphatase activity decreased in the embryos (liver) and females (placenta and liver) by an average of 59 and 42 % (p <0.05), respectively. Beta-glucoronidase also decreased in the liver of the embryo by 26 % (p <0.05).

Neuraminic acid increased in the liver of the females and embryos by 28 and 22 % (p = <0.05), respectively with a corresponding significant rise in serum concentration (see table 6).

A correlation with an increase in overall embryo mortality and enzyme activity was found. The decrease in beta-glucoronidase in the fetal liver and in MDH in the maternal liver correlated with embryo mortality, r -0.67 (p <0.05) and r -0.63 (p <0.05), respectively. In addition, the increase in N-acetyl-neuraminic acid in maternal liver and serum also correlated with embryo mortality, r +0.067 (p <0.05) and r +0.94 (p <0.05), respectively. Thus, changes in the activity of marker enzymes of lysosomes, mitochondria and endoplasmic reticulum of the most important organs and systems reflect embryo mortality.

Table 4. The activity of enzymes of the subcellular organelles and the concentration of N-acetylneuraminic acid in the liver and fetuses and various organs and the blood and serum of the pregnant females in the presence of an embryotoxic effect of formaldehyde.

Organ

Group

of

animals

Malate

dehydrogenase

Glutame

dehydrogenase

Succinate

dehydrogenase

ATPase

B-gluco-

ronidase

Inosine

diphosphatase

N-acetyl

neuraminic

acid

(mg%)
Blood

Serum

Control

Exp.

1.7+0.64

1.35+0.19

0.35+0.04

0.37+0.03

—-

—-

—-

—-

4.84+0.16

7.70+0.38*

0.066+0.01

0.064+0.006

141+4.0

168+6.0*
Liver of

female

Control

Exp.

42.3+3.3

29.6+1.41*

3.1+0.3

4.32+0.1*

9.70+2.27

4.68+0.72*

1.57+0.08

3.08+0.14*

0.96+0.08

0.88+0.12

18.73+1.35

10.78+0.73*

96.2+2.0

124+4.0*
Liver of

fetus

Control

Exp.

13.5+1.4

10.6+0.54*

3.12+0.17

4.53+0.12*

0.91+0.30

1.8+0.30*

3.15+0.17

6.72+0.67*

0.25+0.009

0.16+0.02*

3.36+0.22

1.38+0.30*

154+8.0

188+6.0*
Placenta

Control

Exp.

11.7+0.57

8.4+1.0*

0.27+0.05

0.72+0.14*

1.02+0.15

0.52+0.12*

3.56+0.12

7.74+0.52*

0.23+0.02

0.23+0.02

4.07+0.15

2.35+0.15*

153+5.0

154+3.0

Note: The activity of all the enzymes is given in uM/min/g, and on H+-ATPase is given in uM/h/ mg of protein.
Asterisk = reliability with p <0.05.

Effects of Prenatal FA Exposure on Postnatal Organ Morphology and Function

FA was administered orally in an aqueous solution a dose of 0.5mg/Kg daily to female mongrel rats from the 1st through the 21st day of pregnancy.22 The embryotoxicity of FA was assessed by number of live born for each pregnant rat; the number surviving on the 4th and 21st day of life; the number that died by the 21st day of life; the time of appearance of fur; detachment of concha auriculae; time of eye opening; body mass; and organ (liver, lungs, heart, spleen, adrenal gland, thymus) mass. Organ structural indicators were analyzed at the following ages: new born; two weeks (baby rats); two months (male and females); and 4 months (males) for liver, kidney, lungs and lung mononuclear macrophage system (MPS) in broncho-alveolar lavages (BAL). Hepatocyte alteration index (HAI), ploidy, reticuloendothelial system, lymphoid macrophage infiltration, micronecrotic loci, and extramedullary hematpoiesis centers were assessed. Lung capacity was determined morphometrically. The kidney was examined for normal, atrophic and hypertrophied glomeruli.

The liver damage included a decrease in HAI, a retention of extramedullarhy hemopoiesis, an increase of ploidy (up to 16n) and micronecrotic loci. In addition, there was a lymphoid histiocyte infiltration and fibrosis of blood vessels. The alterations were greater in male (2-3 times greater than controls) vs. the female rats. Kidneys showed atrophic glomeruli with a proportional decrease in normal glomeruli. The lungs had a decrease in alveolar macrophages in BAL.

A mitochondrial enzyme (SDH) was decreased in perivascular hepatocytes at birth, 2 weeks and 2- 4 months after treatment. SDH activity was also decreased in the kidneys (tubule and glomeruli) at 2 and 4 months. Acid phosphatase (ACP) activity decreased in the kidney at 2 weeks and 2 months, rebounding in males at 4 months. This occurred despite atrophic glomeruli being increased at 4 months. The activity of LDH was decreased at 4 months in the liver and alveolocytes (BAL cells).

The assessment of embryotoxicity according to generally accepted indices of the development of offspring is not adequate for the evaluation of the effect of FA on fetuses. A more detailed structural and functional study of the offspring during the postnatal period is required.

Embryotoxic Effect of FA at MAC (Russian Maximum Allowable Concentration)

The embryotoxicity of FA and gasoline was compared to controls on the offspring of 137 female white rats with a total of 853 embryos.23 Female rats (180-200 g) were mated, exposed to FA on days 1 through 19 of pregnancy at the maximum allowable concentration (MAC, 0.5mg/m3), in special chambers for 4 hours/day. Gasoline at 3 times the MAC (300 mg/m3) was also introduced in the same manner in a second group of pregnant females. Control pregnant rats were handled in the same manner without FA or gasoline exposure.

No significant effects were observed on corpus lutea, embryo lost before and after implantation, implanted embryos, lengths of humerus and of embryos. Congenital defects included cryptochordism (20.8+7.6% vs. 1.2+1.2%, p <0.05), delay in ossification of hyoid bone, delay in eruption of upper and lower incisors (days 14-15 vs. 12 of life, p<0.01), and decrease in body weight (days 3, 9, 10 and 17 of life, p<0.05)

Analysis of blood (ABS) showed significant hypercapnia with changes in pCO2 and pO2 (Table 5). This attests to a compensated carbon dioxide acidosis in the mothers and embryos. (The authors did not state whether it was venous or arterial blood.)

Table 5. Indices of the gas composition and pH of the blood of females and offspring exposed to vapors of formaldehyde (FA) and gasoline during prenancy (M+m).

Index

FA

Gasoline

Control
pH

Females

7.206+0.15

7.289+0.020

7.322+0.014
Embryos

6.900+0.034

7.032+0.030

6.977+0.016
pCO2,

(mm Hg)

Females

54.10+2.95b

41.00+1.07

42.91+1.66
Embryos

130.48+5.55b

86.53+4.01a

100.91+3.60
pO2,

(mm Hg)

Females

67.90+5.62

45.75+1.98

51.84+3.37
Embryos

1.23+0.49a

1.30+0.43a

8.16+2.25

Note: Differences from the control are reliable: a = p <0.05; b = p <0.01

Open filed tests demonstrated an increase in motor activity (number of squares visited), increases in frequency of standing and the appearance of emotion (defecation and urination, p <0.01) in 40-day old rats (Table 6). Motor hyperactivity was also present. In sexually mature rats there was an increase in search activity in days 2 through 5 of training. The rate of appearance of motor reflexes did not differ between the exposed and controls.

Table 6. Indices of “open field” behavior of young offspring after prenatal exposure to formaldehyde and gasoline at different testing times (M+m).

Index

Formaldehyde

Gasoline

Control
#. of squares

1st day
85+6

105+6b

76+7
2nd day
45+6b

33+3a

21+4
3rd day
57+4b

23+2a

13+2
# Instances standing
1st day
9.4+0.7

15.2+0.9b

10.8+1.0
2nd day
5.0+0.7a

2.7+0.3

2.4+0.5
3rd
4.3+0.6b

2.6+0.3

1.1+0.3
# of defecations

and urinations

1st day
7.5+0.8b

6.7+0.8b

3.4+0.8
2nd day
6.8+0.8a

6.3+0.6

4.3+0.8
3rd day
7.4+0.7b

5.6+0.5

4.4+0.8
# of washing

movements

1st day
3.1+0.3

1.5+0.2b

2.9+0.5
2nd day
1.4+0.2

0.2+0.1b

1.6+0.4
3rd day
1.0+0.2

0.3+0.1a

0.8+0.2
Latent time, s:
1st day
3.6+0.4

3.3+0.4

3.8+0.7
2nd day
3.9+0.6

0.5+0.2

2.5+1.8
3rd day
1.1+0.3a

0.7+0.2

0.2+0.2

Note: Differences from the control are reliable: a = p<0.05; b = p<0.01

The Effects of FA on the Prenatal Development of Rats with Induced Trace-Element Disorder.

The effect of FA and gasoline inhalation on prenatal development of rats with induced-iron deficiency was reported.24 As rational behind this study the authors stated that the frequency of anemia increases by a factor of 2-3 in women who come in contact with various industrial pollutants in Russia, and iron deficiency anemia is factor in risk of prenatal pathology.

Pregnant white rats (N = 254) with a body weight 180 to 200 g (2511 embryos) were investigated following inhalational exposure to FA (0.5 mg/m3) and gasoline (300 mg/m3) in special chambers administered 4 hrs/day from the 1st to 19th day of pregnancy. The concentrations were controlled by the weight method. Iron deficiency was induced by intraperitoneal by i.p. injection of bipyridyl (chelating agent in 25 % ethanol) at the threshold embryotoxic doses (50 and 40 mg/kg, respectively) in 0.1ml on days 12, 13 and 14 of pregnancy. The control animals were injected with ethanol. The prenatal effect of the xenobiotics was determined by morphological and biochemical methods. Statistical analysis was performed by Silcoxon-Mann-Whitney nonparametric tests. On the 20th day of pregnancy, the females were killed by cervical dislocation. The number of corpora lutea, implantation sites, and live and reabsorbed embryos were counted. The pathology of internal organs and skeleton in embryos were recorded. The average number of metacarpus and metatarsus bone centers per limb was calculated. The acid-base state of the blood of 20-day old embryos and the pregnant females was measured on the ‘Radiometer’ blood microanalyzer (Denmark).

FA caused an increase cryptochordism at 21 %. Bipyridyl caused cleft lip and palate, adhesion and reduction of cartilage of the sacrum and tail in a small number of animals (Table 7). FA inhalation with bipyridyl administration on days 12-14 of pregnancy increased anomalies over controls and FA. There was a significant increase in embryo mortality. In addition, increases in anomalies were observed for cryptochordism (26.7 %), syndactyly (0.4%), adhesion of breastbone (1.2 %) and tail (6.0%), and phoecomelia (1.3%). The overall frequency of developmental defects with combined effects of FA and bipyridyl increased more noticeabley (up to 13.8%) than in the separate effect of FA (6.1%, p <0.01) and bipyridyl (6.6%, p<0.02).

Table 7. Embryonic and teratogenic effects of gasoline (Gas) and formaldehyde (FA) against a background of induced iron trace-element disorder in female rats as of day 12 of pregnancy.

Indices

Control

Gas.

FA

Bipyridyl

Bipyridyl

+ Gas.

Bipyridyl

+ FA
# Preg.

females

29

30

29

18

18

28
Post-implant

Mortality

(4.8+1.3)

(10.1+3.2)

(6.2+1.9)

(12.6+5.5)

(22.7+7.2)

(23.1+)
Anomalies:a
Harelip
0

0

0

1(2.8+2.8)

2(5.2+3.6)

2(5.2+3.6)
Cleft palate
0

0

0

1(2.8+3.6)

2(5.2+3.6)

2(5.2+3.6)
Hydronephrosis
1(0.5+0.5)

3(2.9+1.9)

3(5.1+3.0)

0

0

0
Cryptochordism
0

0

2(20.8+2.6)

5(8.8+4.2)

6(13.5+5.5)

14(26.7+6.1)
Hydrocephaly
0

0

0

0

0

0
Oligodactylia

(fore limbs)

0

0

0

0

0

0
Phocomelia

(hind limbs)

0

0

0

0

0

1(1.3+1.3)
Adhesions:
Breast bone
0

0

0

0

0

2(1.3+0.8)
Digits
0

0

0

0

0

1(0.4+0.4)
Sacral cartilages
0

0

0

1(1.5+1.5)

3(11.7+7.3)

0
Tail cartilages
0

0

0

2(0.7+0.7)

0

5(6.0+2.7)
Partial reduction

of cartilages

0

0

0

0

8(33.7+10.1)

5(9.0+4.6)
# of embryos
221

235

230

196

166

181
Anomaliesb
0

4(1.7+0.8)

14(6.1+1.6)

13(6.6+2.8)

21(12.7+2.6)

39(13.9+2.1)

a = fequency of anomalies per litter; 2 = frequency of anomalies for total embryos.
Percentage of anomalies in parentheses.

The pH of the blood in the mothers and embryos was significantly increased by bipyridyl and decreased by FA and FA + bipyridyl. The pCO2 in the females was increased by FA, while bipyridyl and FA + bipryridyl decreased it. In the embryos there was an increase in the pCO2 by FA and bipyridyl, while the combination of the two decreased this parameter. Changes in the pO2 were also found. In general, the trend was for an increase in this pO2 in both the females and embryos.

A significant decrease was observed in the base reserves of the blood true carbonates and total CO2 in the same embryos with exposure to FA + bipyridyl when compared to the controls, FA and bipyridyl groups. Moreover, there was a significant buildup of acid products of metabolism in embryos with the combined exposure vs. the controls, FA and bipyridyl groups.

Discussion and Analysis of the Papers

The C14FA was distributed to all organs in the adult, the placenta and fetus (Table 1), which was similar to that reported in male F344 rats, guinea pigs and monkeys.25,26 The major difference is that the Japanese demonstrated the incorporation of FA and its metabolites into the placenta and fetus. The quantity of radioactivity remaining in maternal and fetal tissues at 48 hours was 26.9% of the administered dose. The DNA fraction contained 20 % and 50% of total incorporated radioactivity in the maternal and fetal liver at 6 and 24 hours when compared to the acid insoluble fraction (Fig. 1). Of primary interest is that the incorporated radioactivity persisted longer in the fetal liver and brain when compared to the mothers. Also, since FA is a precursor of a number of biological compounds, it would have been of prime interest to determine what fraction resulted from either metabolic incorporation or from chemical reactivity of FA (e.g. crosslinks, adduction, methylation) with biological molecules (DNA, proteins, polypeptide, amino acids, etc.).

FA undergoes addition (adducts and alkylation) and condensation (methene bridges) reactions with proteins and amino acids27 as well as nucleic acids and nucleosides/tides.28 It is a mutagen, crosslinking agent and an immunogen (28-30). Free FA concentrations in the blood are 2.24+0.07 (rats), 1.84+0.15 (Rhesus monkeys) and 2.61+0.14 (humans) ug/g of blood, which did not change following either acute or subchronic inhalation of FA.31,32 Thus, it appears that additional information is required on addition and condensation products of amino acids, polypeptides, nucleoside, etc. of the blood are generated by FA exposure. An increase of N-methyl amino acids would produce endogenous FA, which may have a significant role in mitotic and apoptosis processes. FA generators are responsible for FA formation in tumors and have an impairment of liver antioxidant mechanisms and functional integrity of mitochondria.33-41

FA had adverse effects on zygotes/embryos and bone marrow cells (Tables 2 and 3). The embryos showed cytological injury and high rate of mortality, while bone marrow cells had increased rates of chromosome aberrations and aneuploidy. Similar observations on chromosomes of peripheral lymphocytes have been reported for anatomy and mortuary students.42-44 Classroom exposure to FA at 1.5 to 3.17 mg/m3 was associated with increased frequency of sister chromatid exchanges, aberrations and micronuclei. Concentrations less 1 mg/m3 had no effect on lymphocyte chromosomes, but caused micronuclei in nasal and oral exfoliative cells and changes in lymphocyte subsets (increase in CD19 and decreases in CD4, CD5 and H/S ratio.45,46)

With respect to the effect of FA on embryos additional research is needed. FA is an alkylating agent. Treatment of C3H transplacentally with N-ethyl-N-nitrosourea (alkylating agent) has caused primordial germ cell mutations.47 Also, treatment of female mice within hours after mating with ethyl methanesulfanate, ethyl nitrosourea and ethylene oxide resulted in fetal deaths and malformations.48-51 Thus, further investigation into the zygote/embryonic effects of FA should follow the protocols established for other alkylating agents with attention to the role of potential methyl donors, e.g. N-methyl amino acids.

FA exposure throughout gestation caused a decreased DNA and RNA concentrations, increased weights of bodies and organs (thymus, heart, kidneys and adrenals) and decreased in the weights of lung and liver (Table 3). Microscopy and histochemical observations revealed other abnormalities: involution of lymphoid tissue, numerous extra-medullary hemopoietic centers, decreased glycogen content (myocardium) and liver, decreased AA content of whole fetus and fetal and maternal liver. AA is an antioxidant, produced from glucuronate via the uronic acid pathway, which also is the intermediary route for synthesis of pentoses. The decreased AA content may have resulted from either the utilization of AA as an antioxidant or by interference (inhibition?) of the uronic pathway. It is difficult to interpret the meaning of the decreased DNA and the increased RNA contents of the organs. However, treatment of adult male rats by FA injection was reported to decrease the DNA content of testis and prostate and a decrease of protein content of the prostate and epididymus.52

Cytopathology of organs and alterations of mitochondria, ER and lysosome enzymatic were observed in fetuses following FA inhalation (Table 4). Organ cytopathology included increased ploidy, micronecrotic loci, extramedullary hematopoeitec enters, and degeneration of kidney glomeruli. Concomitant were changes in enzymatic activity of as follows: mitochondria (MDH, SDH, LDH decreased, while GDH increased); ER and lysosomes (ATPase increased while inosine diphosphatase and b-glucorinidase decreased). The impairment lasted in the organs to 4 months of age. In addition, N-acetylneuraminic concentration increased in maternal and fetal tissues. The changes in the enzymatic activity and N-acetyleneuraminic acid correlated with increased fetal mortality. Finally, the development of postnatal behavior was also adversely affected (Table 6).

FA has effects on mitochondrial enzymes, glutathione concentrations and bile production in the liver of many species, including humans.53 FA inhibits the uptake of phosphate by mitochondria ,54,55 and causes the release of GPT, SDH, GSSG and malondialdehyde into the perfusate of isolated livers.56 Intraperitoneal injection results in a 2-fold increase in bile and a significant decrease in glutathione of the liver, lungs and brains.57 An electron microscopic investigation of the perfused isolated livers showed destruction of the mitochondria (ruptured membranes, loss of the cristae) and some damage to the endoplasmic reticulum.56 The protection of the liver from FA toxicity appears to be dependent upon glutathione by formation of the adduct S-hydroxymethylglutathione.58 Thus, the observed effect of FA on mitochondrial and ER functions during embryo/fetal development is also demonstrable in the adult liver.

FA caused preimplantation, prenatal and postnatal abnormalities. The prenatal effects were demonstrable as anomalies and aberrancies in blood buffering capacity with metabolic (formate?) acidosis. The major anomalies were an increased frequency of cryptochordism, a decrease/delay in ossification centers of the hyoid, metacarpus and metatarsal bones, delay in eruption of incisors and a decrease in body weight. Blood pH decreased in the fetus, while the pCO2 (hypercapnia) increased in the fetus and the mother. The true bicarbonates and CO2 were unaffected by FA alone, but increased with iron-deficiency in the fetus and mother. The presence of iron-induced deficiency augmented these abnormalities, along with increased embryo mortality The postnatal effect of FA was tested by maze performance. Open field tests demonstrated an increase in motor activity, increase in standing and appearance of emotion. In sexually mature rats there was an increase in search activity.

FA is metabolized to formate. Alcohols, particularly methanol and ethanol, are metabolized to formate and lactate via an aldehyde. The toxicity of alcohols and formalin in humans and animals includes metabolic acidosis (59-61). Alcohol toxicity generates free radicals, cause an increase in malondialdehyde, and induce lipid peroxidation resulting in DNA single strand breaks (62-66). FA and alcohols probably affect embryos and the fetus via mitochondrial damage. Ethanol and environmental agents trigger apoptotic neurodegeneration in the developing brain (67,68). Oxygen stress, such as that caused by free radical generation, is associated with apoptotic cell death and fragmentation of mitochondrial genome (69-71). Moreover, FA via formaldehyde generators, e.g. alkylating agents, initiates apoptosis (72-74). Mitochondria are the suicide organelles and control apoptosis (75-78). Thus, subtle birth defects (autism, low birth weight, fetal alcohol syndrome, etc.) are probably best understood by investigatiang in utero oxidative stress and mitochondrial damage, rather than by standard FA teratogenic research (79-83).

REFERENCES

1. Occupational exposure to formaldehyde. OSHA Fact Sheet, Jan 1, 1995

2. Formaldehyde, CAS Number 5000. IDLH Documentation. NIOSH, 1996.

3. Feinman SE. (1988) Exposure to Formaldehyde. In: Formaldehyde Sensitivity and Toxicity. CRC Press:Boca Raton, pp. 17-36.

4. Health Effects of Formaldehyde. Environmental Health and Safety, Iowa State University, 2000.

5. Feinman SE. (1988) Skin Effects, Chapers 4-11. In: Formaldehyde Sensitivity and Toxicity. CRC Press: Boca Raton, pp. 49-132.

6. Feinman SE. 1988) Respiratory effects from formaldehyde. In: Formaldehyde Sensitivity and Toxicity. CRC Press:Boca Raton, pp. 135-148.

7. Thrasher JD, Broughton A, Madison R (1990) Immune activation and autoantibodies in humans with long-term exposure to formaldehyde. Arch Environ Health 45:217-223.

8. Formaldehyde, Cas. No. 50-00-0. EPA Health Effects Notebook for Hazardous Air Pollutants. Office of Air Quality Planning and Standard. U.S.E.P.A. 1997

9. U.S. Environmental Protection Agency. Health and Environmental Effects Profile of Formaldehyde. EPA/600/x-85/362. Environmental Criteria and Assessment Office. Cincinnati, OH. 1988

10. World Health Organization. Environmental Health Criteria for Formaldehyde. Vol. 89. World Health Organization, Geneva, Switzerland, 1989.

11. Taskien HK, Kyyronen P, Sallmen M, Virtanen SV, et al. (1999) Reduced fertility among female wood workers exposed to formaldehyde. Am J Ind Med 36:206-212.

12. Taskinen H, Kyyronen P, Hemminki K, Hokkala M, et al. (1994) Laboratory work and pregnancy outcome. J Occup Med 36:311-319

13. John EM, Savitz DA, Shy CM (1994) Spontaneous abortions among cosmetologists. Epidemiology. 5:145-155.

14. Katakura Y, Kishi R, Ikeda T, Miyake H (1990) [Distribution of [14C]-formaldehyde and their metabolites in pregnant mice. Sangyo Igaku 32:42-3.

15. Katakura Y, Okui T, Kishi R, Ikeda T, Miyake H (1991) [Distribution of 14C-formaldehyde in pregnant mice: a study by liquid scintillation counter and binding to DNA. Sangyo Igaku 33:264-65.

16. Katakura Y, Kishi R, Okui T, Ikeda T, Myake H (1993) Distribution of radioactivity from 14C-formaldehyde in pregnant mice and their fetuses. Brit J Ind Med 50:176-82.

17. Kitayeva LV, Kitayeva EM, Pimenova MN (1990) The cytopathic and cytogenetic sequelae of the effect of formaldehyde on female germ cells and bone marrow cells in rats in chronic inhalational exposure. Tsitologia 32:121-26.

18. Gofmekler VA (1968) Effect on embryonic development of benzene and formaldehyde in inhalation experiments. Hyg. Sanit 33:327-32.

19. Pushkina NN, Gofmekler VA. (1968) Changes in content of ascorbic acid and nucleic acids

produced by benzene and formaldehyde. Bull Exper Biol Med 66:868-70.

20. Gofmekler VA, Bonashevskaya TI. (1969) Experimental studies of teratogenic properties

of formaldehyde, based on pathological investigations. Hyg. Sanit 34:266-68.

21. Merkuryeva RV, Litvinov NN, Astakhova LF, Ilin VP, et al. (1996) The significance of changes in the activity of marker enzymes of different intracellular organelles as a criterion in assessing the embryotoxic effect of formaldehyde. Gig Sanit 8:13-15.

22. Belyayeva NN, Zhurkov A, Gaso,pva Kazachkov VI. (1994) The effect of formaldehyd

during the prenatal period on the development of offspring. Gig. Sanit 6:31-34.

23. Senichenkova IN. (1991) The embryotoxic effect of industrial environmental pollutants: Formaldehyde and Gasoline. Gig Sanit 9:35-38.

24. Senichenkova IN, Chebotar NA. (1996) The effects of gasoline and formaldehyde on the prenatal Development of rats with induced iron trace-element disorder. Ontogenz 27:108-13.

25. Heck Hd’A, Chin TY, Schmitz MC (1983) Distribution of [14C] formaldehyde in rats after inhalation exposure. In: Formaldehyde Toxicity, JE. Giibson, ed. Hemisphere Publishing:NewYork, pp.26-37.

26. Jeffcoat AR, Chasalow F, Feldman DB, Marr H. (1983) Disposition of [14C] formaldehyde after topical exposure to rats, guinea pigs, and Monkeys. In: Formaldehyde Toxicity, G.E. Gibson, ed. Hemisphere Publishing:New York, pp. 38-49.

27. French G, Edsall JT. (1945) The reactions of formaldehyde with amino acids and proteins. Protein Chem 2:277-225.

28. Auerbach C, Moustchen-Dahmen M, Moustschen J. (1977) Genetic and cytogenetic effects of formaldehyde and related compounds. Mut Res 39:317-62).

29. Speit G, Schultz P, Merk O. (2000) Induction and repair of formaldehyde-induced DNA-protein crosslinks in repair-deficient human cell lines. Mutagenesis 15:85-90.

30. Carro E, Gasparani S, Gilli G. (1999) Identification of a chemical marker of environmental exposure to formaldehyde. Envir Res 80:132-137.

31. Heck HD, Casanova-Schmitz M, Dodd PB, Schachter EN, et al. (1985) Formaldehyde (CH2O) concentrations in blood of humans and Fischer-344 exposed to CH2O under controlled conditions. Am Ind Hyg Assoc J 46:1-3.

32. Casanova M, Heck HD, Everitt JI, Harrington WW, Popp JA. (1988) Formaldehyde concentrations in the blood of rhesus monkeys after inhalation exposure. Fodd Chem Toxicol 26:715-716.

33. Szende B, Tyihak E, Trezl L, Szoke E, et al. (1988) Formaldehyde generators and capturers as influencing factors of mitotic and apoptotic process. Acta Biol Hung 49:323-329.

34. Kato S, Burke PJ, Fenick DJ, Taajes DJ, et al Mass spectrometric measurement of formaldehyde generated in breast cancer cells upon treatment with anthrcylcine antitumor drugs. Chem Res Toxicol 13:509-516.

35. Spanel P, Smith D, Holland Ta, Singary AL, Elder JB. (1999) Analysis of formaldehyde in the headspace of urine from bladder and prostate cancer patients using selected ion flow tube mass spectrometry. Rapid Commun Mass Spectrum 13:1354-1359.

36. Strubelt O, Dters M, Pentz R, Siegers CP, Younes M (1999) The toxic metabolic effects of 23 aliphatic alcohols in the isolated perfused rat liver. Toxicol Sci 49:133-142.

37. Strubelt O, Younes M, Pentz R, Kuhnel W. (1989) Mechanistic study on formaldehyde-induced hepatotoxicity. J Toxicol Environ Hlthj 27:351-366.

38. Skrzydlewska E, Farbiszewski R. (1997) Decreased antioxidant defense mechanisms after methanol intoxication. Free Radic Res 27:369-375.

39. Skrzdlewska E, Farbiszewski R. (1998) Lipid peroxidation and antioxidant status in the liver, erythrocytes, and serum of rats after methanol intoxication. J Toxicol Environ Health 24:637-649.

40. Szende B, Tyihak E, Szokan G, Katay G. (1995) Possible role of formaldehyde in apoptotic and mitotic effect of 1-methylascrobigen. Pathol Oncol Res 1:38-42.

41. Kalapos MP (1999) A possible evolutionary role of formaldehyde. Exper Mol Med 31:1-4

42. Yager JW, Cohn KL, Spear RC, Fisher JM, Morse L. (1986) Sister chromatid exchanges in lymphocytes of anatomy students exposed to formaldehyde embalming solution. Mutat Res 174:135-139.

43. He JL, Jin LF, Jin HY. (1998) Detection of cytogenetic effects in peripheral lymphocytes of students exposed to formaldehyde with cytokinesis-blocked micronucleus assay. Biomed Environ Sci 11:87-92.

44. Suruda A, Schulte P, Boeniger M, Hayes RB, et al. Cytogenetic effects of formaldehyde exposure in students of mortuary science. Cancer Epidemiol Biormarkers Prev 2:453-560.

45. Ying CJ, Ye XL, Xie H, Yan WS, et al. (1999) Lymphocyte subsets and sister-chromatid exchanges in the students to formaldehyde vapor. Biomed Environ Sci 12:88-94.

46. Vasudeva N, Anand C. (1996) Cytogenetic evaluation of medical students exposure to formaldehyde vapor in the gross anatomy dissection laboratory. J Am Coll Health 44:177-179.

47. Shibuyra T, Muroat T, Horiya N, Matsuda H, Hara T. (1993) The induction of recessive mutations in mouse primordial germ cells with NH-ethyl-M-nitrosourea. Mt Res 290:273-280.

48. Generoso WM, Shourbaji AG, Piegorsch WW, Bishop JB. (1991) Developmental response of zygotes exposed to similar mutagens. Mut Res 250:439-446.

49. Kathoh M, Cadheiro NLA, Cornett CV, Cain KT, et al (1989) Fetal anomalies produced subsequent to treatment of zygotes ethylene oxide or ethyl methanesulfonate are not likely due to the usual genetic causes. Mut Res 210:337-344.

50. Generoso WM, Rutledge JC, Cain KT, Hughes LA, Dowing DH. (1988) Mutagen-induced fetal anomalies and death following treatment of females within hours after mating. Mut Res 199:175-181.

51. Generoso WM, Rutledge JC, Cain KT, Hughes LA, Braden PW. (1987) Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death. Mut Res 176:269-274.

52. Majunder, PK, Kumar VL. (1995) Inhibitory effects of formaldehyde on reproductive system of male rats. Indian J Physio Pharmacol 39:80-82.

53. Beall JR, Ulsamer AG (1984) Formaldehyde and hepatoxicity: a review. J Toxicol Environ Hlth 14:1-21.

54. Tyler DD (1968) The inhibition of phosphate entry into rat liver mitochondria by organic mercurials and by formaldehyde. Biochem J 107:121-123.

55. Fonyo A (1979) Inhibitors of mitochondrial phosphate transport. Pharmacol Ther 7:627-645.

56. Strubelt O, Younes M, Pentz R, Kuhnel W (1989) Mechanistic study on formaldehyde-induced hepatoxicity. J Toxicol Environ Hth 27:351-366.

57. Farooqui MY, Upretti RK, Ahmed AE, Ansari GA (1986) Influence of intraperitoneally administered formaldehyde on bile production and tissue glutathione levels in rats. Res Commun Chem Pathol Pharmacol 53:233-236.

58. Ku RH, Billings RE (1984) Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes. Chem Biol Interact 51:25-36.

59. Tephly TR (1991) The toxicity of methanol. Life Sci 48:1031-1034.

60. Vamvakas S, Teschner M, Bahner U, Heidland A (1998) Alcohol abuse: potential role in Electrolyte disturbances and kidney disease. Clin Nephrol 49:205-213.

61. Pandey CK, Agarwal A, Baronia A, Singh N (2000) Toxicity of formalin and its management. Hum Exper Toxicol 19:330-366.

62. Kadiiska MB, Mason RP (2000) Acute methanol intoxication generates free radicals in rats: ESR spin trapping investigation. Free Radic Biol Med 287:11061114.

63. Strubelt O, Deters M, Pentz R, Siegers CP, Younes M (1999) The toxic and metabolic effects of 23 aliphatic alcohols in the isolated perfused rat liver.

64. Reinke LA, Lai EK, CuBose CM, McCay PB (1987) Reactive free radical generation in vivo in heart and liver of ethanol-fed rats: correlation with radical formation in vitro. Proc Natl Acad Sci 84:9223-9227.

65. Navasumrit P, Ward TH, Dodd NJ, O’Connor PJ (2000) Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure. Carcinogenesis 21:93-99.

66. Nordmann R, Ribiere C, Rouach H (1990) Ehtanol-induced lipid peroxication and oxidative Stress in extrahepatic tissues. Alcohol Alcohol 25:231-237.

67. Ikonomidou C, Bittigau P, Ishimuaru, MJ, Wozniak DF, et al (2000) Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 287:1056-1060.

68. Otney JW, Farber NB, Wozniak DF, Jevtovic-Todorovic V, Ikonomidou C (2000) Environmental agents that have the potential to trigger massive apoptotic neurodengeneration in the developing brain. Environ Health Persp 108(suppl 3):383-388.

69. Yoneda M, Katsumata K, Hayakawa M et al (1995) Oxygen stress induces apoptotic cell death associated with fragmentation of mitochondrial genome. Biochem Biophys Res Comm 209:723-729.

70. Wallace DC, Shoffner JM, Trounce IT et al. (1995) Mitochondrial DNA mutations associated with age and diseases. Biochim Biophys Acta 122271:177-189,

71. Ballinger SC, Couder TB, Davis GS et al (1996) Mitochondrial genome damage associated with cigarette smoking. Cancer Res 56:5692-5697.

72. Szende B, Tyihak E, Trezly L, et al (1998) Formaldehyde generators and capturers as influencing factors of mitotic and apoptotic processes. Acta Biol Hung 49:323-329.

73. Zende B, Tyihak E, Szokan G, Katay G (1995) Possible role of formaldehyde in the apoptotic effect of 1-methyl-ascorbigen. Pathol Oncol Res 1:38-42.

74. Hickman MJ, Samson JD (1999) Role of DNA mismatch repair and p53 induction of apoptosis by aklylating agents. Proc Natl Acad Sci 96:10764-10769.

75. Ferri KG, Kroemer G (2001) Mitochondria – the suicide organelles. Bioessays 23:111-115.

76. Kroemer G (1999) Mitochondrial control of apoptosis: an overview. Biochem Soc Symp 66:1-15.

77. Robertson JD, Orrenius S (2000) Molecular mechanisms of apoptosis induced by cytotoxic agents. Crit Rev Toxicol 305:609-627.

78. Gorman AM, Ceccatelli S, Orrenius S (2000) Role of mitochondria in neuronal apoptosis. Deve Neurosci 22:3480358.

79. Saillenfait AM, Bonnet P, deCeaurriz J (1989) The effects of maternally inhaled

formaldehyde on embryonal and foetal development. Food Chem Toxicol 27:545-548.

80. Martin WJ (1990) A teratology study of inhaled formaldehyde in the rat. Reprod Toxicol 4:237-239.

81. Ulsamer AG, Beall JR, Kang HK, Frazier JA (1984) Overview of health effects of formaldehyde. In: Hazard Assessment of Chemicals. Academic Press:New York, Vol 3, pp. 337-400.

82. U.S. Department of Health and Human Services (1999) Toxicological Profile of Formaldehyde. Agency for Toxic Substances and Disease Registry, Atlanta, GA.

83. Breslin WJ, Liberacki AB, Dittenber DA, Quast JF (1996) Evaluation of the developmental and reproductive toxicity of chlorpyrifos in the rat. Fund Appl Toxicol 29:119-130

84. Hanley TR, Carnew EW Johnson EM (2000) Developmental toxicity studies in rats and rabbits with 3,5,6-trichlor-2-pyrdinol, the major metabolite of chlorpyrifos. Toxicol Sci 53:100-108.

85. Johnson DE, Sceidler FJ, Slotkin TA (1998) Early biochemical detection of delayed neurotoxicity resulting from developmental exposure to chlorpryfos. Brain Res Bull 45:143-147.

86. Lassiter TL, Barone S, Moser VC, Padilla S (1999) Gestational exposure to chlorpyrifos: does response profiles for cholinesterase and carboxlyesterase activity. Toxicol Sci 52:92-100.

87. Chakraborti TK, Farrar JD, Pope CN (1993) Comparative neurochemical and neurobehaviroral effects of repeated chlorpyrifos exposures in young and adult rats. Pharmacol Biochem Behavior 46:219-224).

88. Whitney KD, Seidler Fj, Slotkin TA (1995) Developmental neurotoxicity of chlorpyrifos: cellular mechanisms. Toxcicol Appl Pharm 134:53-62.

89. Chanda SM, Pope CN (1996) Neurochemical and neurobehavioral effects of repeated gestational exposure and exposure to chlorpyrifos in maternal and developing rats. Pharmacol Biochdm Behavior 53:771-776.

90. Dam K, Seidler FJ, Slotkin TA (1998) Developmental neurotoxicity of chlorpyrifos: delalyed targeting of DNA synthesis after repeated administration. Dev Brain Res 108:39-45.

91. Campbell CG, Seidler FJ, Slotkin TA (1997) Chlorpyrifos interferes with cell development in rat brain regions. Brain Res Bull 43:179-189.

92. Xong X, Seidler FJ, Saleh JL, et al. (1997) Cellular mechanisms for developmental toxicity of chlorpyrifos: targeting the adenylyl cyclase signaling cascade. Toxicol Appl Pharm 145:158-174

Stromland K, Miller MT. (1993) Thalidomide embryopathy: revisited 27 years later. Acta Opthalmolo (Copenh). 71:238-24

93. Parman T, Wiley MJ, Wells PG. (1999) Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat Med 5:582-585.

94. Brenner ca, Wolny YM, Barritt JA et al. (1998) Mitochondrial DNA deletion in human oocytes and embryos. Mol Human Repro 4:887-8892.

95. Barritt JA, Brenner CA, Cohen J, et al. (1999) Mitochondrial DNA rearrangements in human oocytes and embrhyos. Mol Human Repro 5:927-933.

96. Thrasher JD. (2000) Are chlorinated pesticides a causation in maternal DNA (mtDNA) mutations? Archiv Enviro Health 55:292-294.

97. Lombard L. (1998) Autism: a mitochondrial disorder? Med Hypotheses 50:497-499.

98. Wallace EC. (1994) Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Scie 91:8730-8746.

99. Giles RE, Blanc H, Cann HM, Wallace DC (1980) Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci 77:6715-6719.

100. Woodbury MA, Zenz C. (1983) Formaldehyde in the home environment: Prenatal and infant exposures. In: Formaldehyde Toxicity (Gibson JE, ed.) Hemisphere Publishing Corp:New York, pp.203-211.