Appendix II
Appendix II
examinations of nervous system structures from 30 suckling Osborne-Mendel rats (“day 1” 5.0 – 8.0 g) and 28 adult rats (150 – 300 g) that were i.p. injected with 50 mg/kg bw acrylamide, 3 times weekly, up to 18 times. A control group was injected with saline. There was a shorter latency for appearance of clinical symptoms in the immature animals (5 or 6 injections) versus adult rats (7 or 8 injections), but symptoms were more severe in the adult animals. [The Expert Panel considers the difference between 5 – 6 and 7 – 8 injections to be of questionable signifi cance.] In the immature animals, slight weakness in hindlimbs became more pronounced and progressed to an inability to stand on hind legs as dosing continued, but some animals showed signs of recovery during the time period while they were still receiving acrylamide injections. Mature animals experienced hindlimb weakness that eventually led to complete paralysis and wasting of hindlimb muscles; weakness persisted for 1 month following the injection period. Axonal and myelin alterations were seen in the younger rats at earlier stages of neuropathy, but the changes became more pronounced and complicated in adult rats. Evidence of neurologic regeneration (e.g., growth cones and axonal sprouts) appeared in immature rats during the injection period, but was not seen in adult rats until 20 days following the last injection. The study authors concluded that young rats are more susceptible to acrylamide but also have a greater ability for regeneration than do adult rats.
Ko et al. (108) examined the effects of age on acrylamide-induced neurotoxicity in male ICR mice in a study conducted at a Taiwanese university. Groups of 3-week-old and 8-week-old mice were given drinking water containing 400 ppm acrylamide. Acrylamide intake was estimated at (mean ± SD) 91.8 ± 20.6 mg/kg bw/day in the 3-week-old mice and 90.8 ± 10.9 mg/kg bw/day in the 8-week-old mice. Control mice for each age group were given drinking water without acrylamide. Time to reach three different stages of neurologic symptoms was recorded in the treated mice. An initial stage was characterized by normal appearance, but decreased performance on rotarod and swelling of motor nerve terminals. Signs observed in the early stage included paraparesis and terminal nerve swelling. Symptoms of the late stage included quadriparesis, denervation, and decreased amplitude of muscle-action potential. Total numbers of mice treated were not specifi ed but 3 – 24 mice/group were examined pathologically during 3 different periods. Each of the stages occurred signifi cantly earlier in the 3-week-old mice than in the 8-week-old mice (mean days ± SD): 7.1 ± 1.1 vs. 15.6 ± 4.0 days, respectively, for the early stage and 15.3 ± 2.1 vs. 31.7 ± 6.0 days, respectively, for the late stage.
The study authors concluded that younger mice are more susceptible to acrylamide-induced toxicity and that pathologic symptoms occur prior to neurologic symptoms.
Husain et al. (109) studied the effects of acrylamide exposure on brain neurotransmitters in developing and mature male Wistar rats in a study conducted at an Indian research center. In the fi rst study, dams were orally administered saline or 25 mg/kg bw/day acrylamide during the entire lactation period. The male offspring of the dams were sacrifi ced at 2, 4, 8, 15, 30, 60, or 90 days of age for an examination of brain neurotransmitter levels. During each time period, brains from 1 to 3 rats were pooled and six observations were made. Compared to rats in the control group, the rats exposed to acrylamide through milk had signifi cantly lower levels of brain noradrenaline and dopamine at 2 – 15 days of age and 5-hydroxytryptamine at 2 – 30 days of age. Monoamine oxidase activity was signifi cantly increased and acetylcholinesterase activity was signifi cantly decreased at 2 – 30 days of age. In a second study, male rats were orally administered saline or 25 mg/kg bw/day acrylamide for 5 days at 12, 15, 21, or 60 days of age. Neurotransmitter levels were measured in various brain regions. It appears that at least six animals per group were examined and it was stated that reported values represented the mean of
Appendix II
fi ve observations. Signifi cant reductions in neurotransmitter levels in treated compared to control rats that were observed only in immature rats (≤21-days-old) included noradrenaline in the pons medulla and basal ganglia and dopamine in the pons medulla. Neurotransmitter levels reduced in all ages of treated compared to control rats included noradrenaline in midbrain; dopamine in cerebellum and midbrain; and 5-hydroxytryptamine in pons medulla, hypothalamus, and cerebral cortex. The study authors concluded that immature rats are more vulnerable to neurotoxicity induced by acrylamide exposure and that effects are localized within certain brain regions.
Due to unpublished observations that younger rats are more susceptible to acrylamide-induced paralysis, Dixit et al. (110) compared hepatic GST activity and glutathione content in young and mature albino rats. The study was conducted at an Indian research center. At the ages of 9, 15, or 26 days, or 4 months, rats were given 50 mg/kg bw/day acrylamide by i.p. injection for 5 days [number of rats in each group not specifi ed]. Control rats were injected with sodium chloride vehicle. One day following the last injection, livers were homogenized for a determination of hepatic GST activity and glutathione content. Generally, GST activity increased with age and acrylamide treatment reduced GST activity in all age groups. The magnitude of GST reduction was greatest in the 15-day-old rats, in which early development of paralysis was reported [neurotoxicity data not shown]. Acrylamide treatment resulted in signifi cant decreases in reduced and oxidized glutathione content only in the 26-day-old rats. [Methods of statistical analysis were not discussed for any of the data reported in this study.]
[The Expert Panel fi nds the literature on age-related susceptibility to be diffi cult to interpret, with some studies showing greater susceptibility in young animals and others not showing such differences in susceptibility.]
2.5.2 Ontogeny, polymorphism, and other factors affecting metabolism 2.5.2.1 GST and glutathione
As noted in Section 2.1.3, a major pathway of acrylamide biotransformation is conjugation with glutathione, catalyzed by GST. Cytosolic GST is a family of soluble dimeric enzymes consisting of 13 different subunits from 5 different subclasses (111):
1. alpha (GSTA1, GSTA2, GSTA3, GSTA4)
2. mu (GSTM1, GSTM2, GSTM3, GSTM4, GSTM5) 3. pi (GSTP1)
4. theta (GSTT1, GSTT2) 5. sigma (GSTZ1)
Subunits can dimerize only with members of the same class to form homodimers or heterodimers (111).
The alpha, mu, and pi enzymes are the most abundant GST classes in mammalian species (112).
McCarver and Hines (111) reviewed the ontogeny of GST and other phase II metabolizing enzymes.
Limited information is available on the ontogeny of human hepatic GST. The ontogeny of human hepatic GST is summarized in Table 19. GSTA1 and GSTA2 are expressed in the fetus and expression reaches adult levels at 1 – 2 years of age, following a 1.5 – 4-fold increase. Low levels of GSTM are also detected in the fetus, and expression increases 5-fold at birth to reach adult levels. GSTP1 expression
Appendix II
is highest in 10 – 22-week-old fetuses and decreases during the second and third trimesters; GSTP1 is expressed in neonates but not adults. McCarver and Hines (111) concluded that though limited, the information on GST ontogeny demonstrates that substantial changes occur during development. Such changes can affect chemical disposition and clinical outcomes, which also depend upon the balance of other phase I and phase II enzymes. Currently, the information is inadequate to predict adverse reactions or determine appropriate therapies for fetuses, neonates, infants, and children.
Table 19. Ontogeny of Human Hepatic GST, McCarver and Hines (111).
Gene Prenatal Trimester
Neonate 1 Month-
1 Year 1-10
Years Adult
1 2 3
GSTA1/A2 + + + + + + +
GSTM + + + + + + +
GSTP1 + + + + – – –
GST polymorphisms are recognized, most notably null phenotypes for the GSTM1 (GSTM1*0) and GSTT1 (GSTT1*0) alleles (112). No gene products are expressed in individuals who are homozygous for the null phenotype. Frequency of GSTM1*0 homozygosity is 58% in Chinese, 52% in English, 48% in Japanese, 43% in French, and 22% in Nigerian individuals (112). Frequency of GSTT1*0 homozygosity is 16% in English, 12% in German, 38% in Nigerian, and 32% in West Indian individuals.
Friedman (11) noted that reductions in glutathione levels can increase susceptibility to acrylamide-induced toxicity by leaving cell membranes less protected against oxidative stress. Factors that can reduce glutathione levels include diets low in the amino acids cystine and methionine; oxidative stress; and liver damage.
2.5.2.2 Cytochrome P450
As noted in Section 2.1.3, a second major pathway of acrylamide biotransformation is oxidation to glycidamide through cytochrome P450. CYP2E1 was identifi ed as the enzyme responsible for biotransformation of acrylamide in mice and rats, but no information on the specifi c enzyme in humans could be located. [The Expert Panel fi nds it reasonable to suspect that CPY2E1 is the isoform responsible for acrylamide biotransformation in humans.] Hines and McCarver (113) noted that fetal liver contains 30 – 60% of total adult cytochrome P450 content; levels continue to increase at birth and reach adult levels at 10 years of age. However, signifi cant expression differences are noted among individual P450 enzymes. Anzenbacher and Anzenbacherova (114) noted that polymorphisms among cytochrome P450 enzymes could result in defective, qualitatively different, reduced, or enhanced activities of the enzymes. Enzyme activities could also be affected by factors that induce or inhibit enzymes, including diet, age, and health. Calleman et al. (28) noted that mixed results were found in animal studies examining the effects of inducers or inhibitors of oxidative metabolism on acrylamide-induced neurotoxicity. Calleman noted that some inducers or inhibitors can impact other metabolic enzymes, including GST.
2.5.3 Gender-related differences
There is no indication that males or females are more susceptible to general toxicity induced by
Appendix II
acrylamide exposure. Acrylamide has adverse effects on the reproductive systems of males, but not females, as discussed in Section 4.
2.6. Summary of General Toxicology and Biologic Effects