P.1 

OAR Box 1126 

Prepped by Ollie Stewart 

Document Number: 

31)11-1-153 

Docket Number: 

A-83-22 

Printed 4/18/2008 9:58:22 AM Sheet 31 of 182 
P.2 

Toxic Effects of Lead of Neuronal Development and Functioi 

Robert Freedman, Lars Olson, and Barry J. Hoffer 

B ie tn f i 

YM I 3 1932 

EPA AIR DOCKET 

Departments of Psychiatry and Pharmacology, Denver VAMC

 L~a'rib' uhiversity ot 

Colorado Health Sciences Center, Denver, CO 80262 and Department of Histology, 

Karolinska Institute, Stockholm, Sweden 

The effects of lead on the development of the nervous system are of 

immediate concern to human welfare. While it is clear that lead can affect 

neuronal development at levels of exposure within the range found in the 

environment, the particular mechanism of the disruption is not easily 

ascertained. Lack of knowledge of the mecrianism of damage hampers i ts 

treatment and prevention. The purpose of our research is to develop a model 

system in which the effects of lead on central nervous.system development can 

be demonstrated. The complexity of the brain is a barrier to such 

investigations, because often it is not clear if apparent toxic effects 

represent changes secondary to somatic changes, such as endocrine or 

hematological defects that could affect brain development, or even 

transneuronal effects caused by toxicity at a distal site that deprive a brain 

area of a synaptic input needed for its proper development. A related 
P.3 

problem is tha redundancy of compensatory systems in ths brain. Such systems 

may disguise the severity of ths initial toxic insult and themselves can cause 

functional disturbances. We have grafted discrete brain regions derived from 

rat fetuses into ths anterior chamber of the eye of adult hosts in an effort 

to study neuronal development in a system that minimizes such difficulties. 

The brain pieces continue organotypic development in the eye, but are isolated 

from possible problems in the development of the endocrine and other somatic 

systems, because the adult host has these systems already fully developed. 

Similarly, toxic effects mediated by connecting brain areas are eliminated 

since tha transplant is isolated in ths anterior chamber of the eye. using 

this system ws have discovered that lead induces a hypernoradrensrgic 

innervation of central nsrvous system tissue. Ths increased innarvation is 

observed not only structurally, but also functionally. Since norepinephrine 

is an inhibitory neurotransmitter, ths ingrowth results in a slowing of the 

discharge of nsurons that normally receive noradrensgic innervation. Studies 

of othsr tissues suggest that increased axonal growth and innervation may be a 

gansral problem of lead intoxication that extends beyond the central 

adrenergic system to encompass other brain areas, as well as peripheral 

sympathsfcic systems. Hyperactivity might be ths behavioral consequence of 

thssa alterations in nsuronal dsvelopment. 
P.4 

Fto. I. Examples of intraocular brain grafts as observed In vivo at approximately lOx magnification: (a) Hippocampal graft obtained from a fetus with a CRL of 25 mm 6 months postgnftlog. The host 

animal was maintained on I * lead acetate. These grafts show normal vascularization (several blood 

vessels can be seen oo graft surface) but slightly impaired volume increase as compared to controls, (b) 

Oraft of the developing substantia nigra area obtained from a fetus with a CRL of 24 mm 2 months after 

grafting to host animal on tap water. In contrast to cortical grafts such as in (a), these brain stem grafts 

obtain a final volume smaller than at grafting. White area in upper part is light reflex. 

•ayaa 

1-

err T " 

J 

si 

> •> «*Ma e 

Fio. 2. Lead levels in blood (solid line, Mg/liter) snd 

brain IH situ (dotted line, mg/kg) as a function of varying duration of exposure to 1% lead acetate in drinking 

water. Points show mean values, and bars, SEM. 
P.5 

CORTEX CEREBEUI 

tNIr 

tat. *• 03RTEX CSEBail 

r T * WB8 '—\ . . i . i >*«S 

frioji Effects of chronic 1% lead aceute on growth ofthe cerebellar anlage In oeulo. Cerebellar 

grafts were taken from fetuses with a CRL of 11-12 mm corresponding to gestational Days 14 (a), 

and 15-17. gestational Days 16-17 (b). Grafts from more immature donors grew to larger final sizes 

(b). There were no significant effects of lead on growth in either of Ihe two groups, n » 19-24 (a) and 

21-28 (b). 
P.6 

Pioutl 1. Creeyi-violet-etained section of a lead-treated cerebellar 

graft obtained from day 14 of gestation aa seen after intnoeoiar 

maturation. Note the high degree of organotypic organisation 

Two fobs of cerebellar cortex can be seen with free surfaces eovered by plal membrane. 
P.7 

fTmW^J} 

100 m sec 

B. "*• 

100-

« 76-

o so: 

75 msec 

C. 

so aec 

FIGURE 2. Spontaneous discharge from a graft Purkinje cell in a sodium acetate-treated animal (A) Action potential record phol 

from the oscilloscope, (fl) Interspace Interval histogram with prominent model peak indicating regularity of discharge. AbedsM • 

is for full scale. (ORatemeter record again showing sustained regular discharge. 

phed 

I I *-*•* Aeeiato 

H Sodlua Aoetate 

FIOUBI S. Histogram showing the distribution of spontaneous discharge rates of Puridnje neurons from lead acetate-treated and 

sodium acetate-treated cerebellar grafts. There is a marked difference between the two groups which is statistically significant 

(p< 0.001). Nota that the majority of ceUs encountered in leadtreated grafts were suenL (•) denotes cells which were aU derived 

from' one graft, the only lead-treated graft containing Purkmje 

neurons with sustained, spontaneous discharge. 
P.8 

• • 

non-Injected 1 mo/Kg 

Control* PbAe 

Smg/Kfl Malnourished Untreated 

PbAe Control 

FIGURE 4. Bar graph ahowing mean discharge rate ± SEM of cerebellar Purkmjo neurone from animals injected postnatally with NaAc, 1 

mg PbAc/kg, 8 mg PbAc/kg, and nonmjoctcd controls. In addition, the mean firing rates rf Purkinje Morons from malnourished animals 

and from animals concomitantly raised in normalized litters are illustrated. The rates in 8 mg PbAe/kg animals aro significantly different 

from those of NaAc animals (p < 0.01). 

J 
P.9 

FIGURE 6. Effecto of kad Injected into Ae anterior chamber of the eye in a 8-pL volume on sympathatls odreaargis narvee in the rot Iris. 

Illustrated ore fiuoreccenee mJa-ophotographs from representative areas of iris whole mounts processed for vtotAntlon of catwhoHaminea 

neeordihg to Fakk and HiOarp. (/USliam-lnJected eye. Normal density of adrenergic ground plexus in dilate? oras of the irio. » 114. 

(fl) Adraitargie hyperinnervatke U days after injection of lead acetate. There ia almost no decreaca in msoressisncQ intensity (Le., cotoehohinlfts content) cf individual nerve fibers. An Innervated blood vessel traverses from upper loft to lower right, x 114. 
P.10 

• No Acetate Control 

Pb Acetate • firontal Cortex 

I20-, 

Pb Acetate-Parietal Cor ten 

Pb Acetate-Occipital Cortex 

*) ps.Oft 

• P» Ol 

FIGURE 8. Cortical noradrenergic varieoeitiea in animals treated 

neonatally with lead- or sodium aceute. Estimates were made by 

blind observers using Falck-HUlarp fluorescence histochemical 

techniques. Note elevation in lead-treated animals in all three 

cortical regions.